Inhibition of LRRK2 Ameliorates Aspergillus fumigatus Keratitis by Regulating STING Signaling Pathways

Abstract

Purpose: The purpose of this study was to investigate the role of LRRK2 in the inflammatory response to fungal keratitis (FK) and elucidate the underlying mechanisms.

Methods: The protein levels of leucine-rich repeat kinase 2 (LRRK2), p-LRRK2, and stimulator of interferon genes (STING)-related proteins were assessed by western blot analysis. ELISA and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to evaluate the inflammatory response induced by Aspergillus fumigatus. Mass spectrometry was performed to identify the interaction partners of LRRK2. The glutathione S-transferase (GST) pull-down assay and co-immunoprecipitation (co-IP) were used to verify the interaction between LRRK2 and STING. Additionally, fungal load determinations and clinical score assessments were conducted to determine corneal infection in a mouse model.

Results: A. fumigatus stimulation promoted the phosphorylation of LRRK2 through Toll-like receptor 2 (TLR2) in human corneal epithelial cells (HCECs) and mouse corneas. LRRK2 overexpression enhanced the A. fumigatus–induced inflammatory response, and LRRK2 knockdown alleviated A. fumigatus keratitis both in vitro and in vivo. Mass spectrometry identified STING as a novel interaction partner of LRRK2. Moreover, A. fumigatus treatment enhanced the interaction between LRRK2 and STING, resulting in the phosphorylation and activation of STING. The phosphorylated STING then triggered its downstream signaling pathways, exacerbating the severity of A. fumigatus keratitis. LRRK2 inhibitor (LRRK2-IN-1) significantly mitigated the inflammatory response and corneal damage caused by A. fumigatus stimulation.

Conclusions: LRRK2 inhibition ameliorates A. fumigatus–induced inflammation through modulating STING signaling pathways in both HCECs and mouse models. Our results suggest that targeted inhibition of LRRK2 could be a promising strategy for FK treatment.

Fungal keratitis (FK) is a severe corneal infection caused by pathogenic fungi, often leading to significant vision impairment or blindness if not treated promptly.¹ The infection is commonly caused by saprophytic fungal pathogens, including Aspergillus and Fusarium species.² The incidence of FK is rising due to factors such as excessive corticosteroid application, previous corneal injury, and misuse of contact lenses, especially in developing countries.³ Current treatment options typically involve topical antifungal agents such as natamycin and voriconazole. However, these treatments face challenges such as inadequate ocular penetration, low bioavailability, and the growing issue of drug resistance, all of which contribute to poor treatment outcomes.⁴ Such challenges highlight the urgent need to develop new and effective antifungal drugs to combat antimicrobial drug resistance and enhance treatment efficacy for FK. 

Corneal epithelial cells (CECs) form the outermost layer of the cornea, playing a crucial role in maintaining a refractive surface essential for vision and ocular tissue protection.⁵ As the first line of defense against fungal invasion, CECs are essential for preventing and controlling infections. Damage to these cells facilitates deeper fungal penetration, triggering more severe inflammatory responses.⁶ This progression can result in increased corneal opacity, neovascularization, corneal ulcer formation, reduced visual acuity, and even vision loss. When facing invading fungi, the pattern recognition receptors (PRRs) of CECs can recognize conserved structures from microorganisms referred to as pathogen-associated molecular patterns (PAMPs).⁷ Upon binding of PAMPs to PRRs, innate immune responses are triggered through the activation of various signaling pathways, which lead to fungal internalization through the production of pro-inflammatory cytokines, including IL-1β, IL-6, IL-8, TNF-α, and LL37.⁸ Appropriate inflammatory responses help the body eliminate pathogens, but excessive inflammation can lead to immunopathological tissue damage. Therefore, precise regulation of inflammation is crucial in antimicrobial immunity. 

Leucine-rich repeat kinase 2 (LRRK2) is a serine/threonine kinase that belongs to the Roco protein family.⁹ Initially, LRRK2 was identified as a factor associated with familial and idiopathic Parkinson's disease (PD) risk.¹⁰ Recently, high-resolution structures of full-length human LRRK2 in various functional states have been elucidated, offering a structural basis for the development of LRRK2-targeted therapeutic interventions.¹¹ Additionally, several LRRK2 kinase inhibitors have been designed and developed, many of which are currently undergoing clinical trials, thus providing valuable tools for investigating the functional role of LRRK2.^12,13 Beyond its association with PD, LRRK2 has garnered attention for its crucial role in immune regulation and inflammatory response, positioning it as a potential therapeutic target for inflammatory diseases.¹⁴ The LRRK2 G2019S mutation, characterized by increased kinase activity, has been shown to enhance immune response pathways and exacerbate airway hyperresponsiveness in mouse models of asthma.¹⁵ LRRK2 promotes inflammatory cytokine induction by enhancing the signaling strength of the Nod2–Rip2 signaling pathway in macrophages under endoplasmic reticulum stress.¹⁶ In animal models of colitis, transgenic mice overexpressing LRRK2 exhibit more severe colitis induced by dextran sodium sulfate (DSS). Pharmacological inhibition of LRRK2 function may potentially ameliorate colitis in patients with inflammatory bowel disease.¹⁷ Furthermore, multiple studies have indicated a relationship between LRRK2 and infections, particularly with bacterial pathogens.¹⁸ Early research found that LRRK2 contributes to the restriction of intestinal pathogens such as Salmonella in macrophages, with inhibition of LRRK2 kinase activity leading to increased susceptibility to Salmonella infection in mice.^19,20 LRRK2 also regulates the innate immune response to Mycobacterium tuberculosis by maintaining mitochondrial homeostasis.²¹ Loss of LRRK2 kinase activity suppresses the proliferation of M. tuberculosis in mouse and human macrophages.²² However, the precise role of LRRK2 in FK remains unclear. 

In this study, we investigated the role of LRRK2 in the inflammatory response to FK and elucidate the underlying mechanisms. We demonstrated that LRRK2 inhibition alleviates Aspergillus fumigatus–induced inflammation in both HCECs and mouse models. Additionally, we determined that LRRK2 regulates A. fumigatus keratitis through interaction with STING and modulation of its downstream signaling pathways. Our findings provide novel insights into the mechanisms of inflammatory responses in A. fumigatus keratitis and suggest that targeted inhibition of LRRK2 could be a promising strategy for managing this condition. 

Materials and Methods

Cell Culture

The SV40-immortalized human corneal epithelial cell (HCEC) line, kindly provided by Fu-Shin Yu, PhD, at Wayne State University (Detroit, MI, USA), was cultured in Gibco Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient Mixture F-12 (11320033; Thermo Fisher Scientific, Waltham, MA, USA). The medium was supplemented with 50% Gibco Defined Keratinocyte Serum-Free Medium (K-SFM, 10744019; Thermo Fisher Scientific) and 1% Gibco penicillin/streptomycin (15070063; Thermo Fisher Scientific). The HEK293T cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in high-glucose Gibco DMEM (11965092; Thermo Fisher Scientific) containing 10% fetal bovine serum. Authentication of the cells was performed using short tandem repeat profiling. The cells were maintained at 37°C in a humidified incubator with 5% CO₂. 

A. fumigatus Preparation

A. fumigatus (strain 93024) was sourced from the China Center for Type Culture Collection (CCTCC, Beijing, China). Conidia and hyphae were prepared as described previously.²³ Briefly, after 7 days of incubation on Sabouraud dextrose agar (213400; BD Bioscience, Franklin Lakes, NJ, USA) at 37°C, conidia and hyphae were harvested and cultured in liquid Sabouraud medium with shaking at 500 rpm for 18 hours. The hyphae were then ground into 20- to 40-µm fragments, washed with PBS, and centrifuged. For in vitro experiments, hyphae were heat inactivated at 56°C for 60 minutes and used at 1 × 10⁶ hyphal fragments/mL. The multiplicity of infection between fungi and cells was 5. For in vivo studies, live hyphae were diluted in PBS to a final concentration of 1 × 10⁸ hyphal fragments/mL. 

FK Mouse Model Construction

All animal experiments were approved by the Ethics Committee of Qilu Hospital of Shandong University and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The male C57BL/6 mice, 7 to 8 weeks old, were purchased from Gempharmatech Co., Ltd. (Nanjing, China). After the mice were anesthetized with pentobarbital, the central corneal area was scraped using a 26-gauge needle. The ocular surface was then coated with 5 µL of a hyphae solution, and the cornea was covered with a molded artificial parafilm. An additional 5 µL of the hyphae solution was introduced into the gap between the parafilm and the cornea after the eyelids were sutured together. Twelve hours after molding, the parafilm was removed, and corneal examinations were conducted. The clinical score was determined according to the criteria established by Wu et al.²⁴ 

Quantitative Real-Time PCR

Total HCEC RNA was isolated with Invitrogen TRIzol reagent (15596018; Thermo Fisher Scientific), and corneal RNA was purified using the RNeasy Micro Kit (74004; QIAGEN, Hilden, Germany) following the manufacturer's protocol. RNA was converted to cDNA via reverse transcription with the PrimeScript RT Reagent Kit (RR037A, Takara Bio, Shiga, Japan). Real-time PCR was conducted using SYBR Premix Ex Taq (RR420A; Takara Bio) on the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primer sequences are listed in Supplementary Table S1. Relative gene expression levels were calculated using the 2^−ΔΔCt method, with normalization to β-actin. 

Antibodies and Reagents

Antibodies against Toll-like receptor 2 (TLR2; 66645-1-Ig, 1:1000), β-actin (20536-1-AP, 1:50,000), IκBα (10268-1-AP, 1:1000), glutathione S-transferase (GST; 10000-0-AP, 1:1000), MYC (16286-1-AP, 1:1000), hemagglutinin (HA; 81290-1-RR, 1:1000), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60004-1-Ig, 1:5000), lamin B1 (12987-1-AP, 1:2000), STING (19851-1-AP, 1:2000), interferon regulatory factor 3 (IRF3; 11312-1-AP, 1:1000), and TANK-binding kinase 1 (TBK1; 28397-1-AP, 1:1000) were purchased from Proteintech (Wuhan, China). Antibodies against p-STING (Ser366, 19781; Ser365, 72971; 1:1000), p-TBK1 (5483, 1:1000), p-IκBα (2859, 1:1000), p-IRF3 (4947, 1:1000), p-p65 (3033, 1:1000), and p65 (8242, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against LRRK2 (ab133518, 1:1000) and p-LRRK2 (Ser935, ab133450; 1:1000) were purchased from Abcam (Cambridge, UK). Invitrogen Goat anti-Rabbit IgG Alexa Fluor 594 (A-11012, 1:200) for immunofluorescence staining was obtained from Thermo Fisher Scientific. LRRK2-IN-1 (S7584) and C176 (S6575) were acquired from Selleck Chemicals (Houston, TX, USA). 

RNA Interference

The human LRRK2, TLR2, and STING small interfering RNAs (siRNAs) were designed and synthesized by GenePharma (Shanghai, China), and mouse LRRK2 siRNA (sc-45750) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). HCECs were seeded in 6-cm dishes and cultured overnight. The following day, the cells were transfected with 80-nM siRNA or negative control (NC) siRNA for 24 hours using Invitrogen Lipofectamine 2000 Reagent (11668-019; Thermo Fisher Scientific). For in vivo assays, a subconjunctival injection of LRRK2 siRNA was administered to the cornea prior to A. fumigatus infection. The siRNA sequences are detailed in Supplementary Table S2. 

Plasmid Transfection

The HA–LRRK2 and Myc–STING vectors were constructed by inserting the coding sequences into pcDNA3.1 vectors (Addgene, Watertown, MA, USA). Cells were plated in 6-cm dishes and cultured overnight. The following day, the cells were transfected with 4 µg of plasmid using Lipofectamine 2000 Reagent, and 24 hours after transfection cells were treated with A. fumigatus. 

Western Blot

The western blot assay was performed as described previously.²⁵ In brief, total protein was extracted from mouse corneas or HCECs using radioimmunoprecipitation assay (RIPA) lysis buffer (P0013K; Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (P1005; Beyotime). Protein concentration was measured using a bicinchoninic acid (BCA) assay (P0010S; Beyotime). Protein samples were resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes (MilliporeSigma, Burlington, MA, USA). The membranes were first blocked with non-fat milk and then incubated with primary antibodies, followed by incubation with secondary antibodies. Protein bands were imaged with an Amersham Imager 600 (GE Healthcare Technologies, Chicago, IL, USA). Quantification of protein bands was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA), with β-actin as the endogenous control. 

ELISA Analysis

HCEC supernatant was collected and centrifuged for 5 minutes at 1000g. Mouse corneal homogenate was prepared by ultrasonic disruption in PBS containing a protease inhibitor cocktail. The homogenate was then centrifuged, and the supernatant was collected. The levels of IL-1β, IL-6, and TNF-α were measured using specific ELISA kits, following the manufacturer's instructions. Optical density (OD) values were recorded at 450 nm using a microplate reader. Details of the ELISA kits used in this study are listed in Supplementary Table S3. 

Immunofluorescence Staining

HCECs were plated in glass-bottom dishes (NEST Biotech, Wuxi, China) and subjected to specified treatments. Cells were fixed with 4% paraformaldehyde for 15 minutes, blocked with 2% bovine serum albumin for 30 minutes, and then incubated with primary antibody (1:100) overnight. The following day, cells were stained with the corresponding secondary antibody (1:200) for 1 hour, and 4′,6-diamidino-2-phenylindole (DAPI) was used to label nuclei. Images were captured using a fluorescent microscope (X81; Olympus Optical, Tokyo, Japan). 

Nuclear and Cytoplasmic Protein Extraction

Nuclear and cytoplasmic proteins were separated using the Nuclear and Cytoplasmic Protein Extraction Kit (P0027; Beyotime), following the manufacturer's instructions. 

Fungal Load Determination

Infected corneas were homogenized using a micro tissue grinder in PBS. A 30-µL aliquot of the corneal homogenate was plated onto Sabouraud dextrose agar plates and incubated for 24 hours at 37°C to allow fungal colonies to develop. The number of colony forming units was then counted. 

GST Pull-Down Assay

The coding sequences were cloned into the pGEX-4T-1 and pET-22b(+) vectors to produce GST–STING and His–LRRK2 constructs. The protein expression was induced with 1-mM isopropyl β-d-1-thiogalactopyranoside (IPTG) in Escherichia coli BL21. The proteins were purified using Beyotime Protein Purification Kits. For the GST pull-down assay, GST–STING, His–LRRK2, and GST–tag Purification Resins (P2253, Beyotime) were incubated together for 2 hours in the protein binding buffer. The GST–tag Purification Resins were then washed twice with washing buffer, boiled in SDS loading buffer, and analyzed by western blot. 

Co-Immunoprecipitation

Cells were lysed using Western and IP Lysis Buffer (P0013B; Beyotime) supplemented with protease inhibitor cocktail. The cellular supernatants were collected after centrifugation. Subsequently, 800 µg of the cellular extract was incubated overnight with 5 µL of primary antibodies on a rotary shaker. The mixture was then incubated with protein A/G magnetic beads (B23202; Selleck Chemicals, Houston, TX, USA) for 2 hours at 4°C. Following three washes with lysis buffer, the bound immune complexes were eluted by boiling in SDS loading buffer and analyzed by western blot. To prevent interference from denatured IgG, anti-HA Nanobody Magarose Beads (KTSM1335; AlpaLifeBio, Shenzhen, China), anti-Myc Beads (KTSM1336; AlpaLifeBio), and HRP-Conjugated Mouse anti-Rabbit IgG Light Chain (AS061; ABclonal, Wuhan, China) were used. 

Mass Spectrometry Assay

HCECs transfected with either pcDNA3.1 (HA) or pcDNA3.1 LRRK2 (HA-LRRK2) were lysed using Western and IP Lysis Buffer. Following centrifugation, the cleared lysates were incubated overnight with anti-HA Nanobody Magarose Beads. The beads were then washed three times with lysis buffer and denatured with SDS loading buffer. The loading buffer with immunoprecipitated proteins was collected and separated by SDS-PAGE, followed by silver staining to visualize the protein bands. Gel slices with the most significant differences in band intensity were excised and sent for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis by APTBIO (Shanghai, China) to identify the proteins. 

In Vitro Kinase Assay

A reaction mixture (50 µL) containing reaction buffer (25-mM Tris, pH 7.5; 5-mM β-glycerophosphate; 2-mM dithiothreitol; 0.1-mM Na₃VO₄; 10-mM MgCl₂), 200-µM adenosine triphosphate (ATP), 25 ng His–LRRK2 protein, and 40 ng GST–STING was incubated at 30°C for 30 minutes. Phosphorylation of STING was detected by immunoblotting with the p-STING (Ser366) antibodies. 

Minimum Inhibitory Concentration Assay

The minimum inhibitory concentration assay was performed as described previously.³ The LRRK2-IN-1 was diluted to concentrations of 0, 1, 5, 10, 20, 40, and 80 µg/mL using Sabouraud liquid medium. A. fumigatus spores were added to the medium at a final concentration of 1 × 10⁶ hyphal fragments/mL. Then, 100 µL of medium was cultured in 96-well plate for 24 hours at 37°C. OD values were measured at 540 nm. 

Statistical Analysis

Values are expressed as mean ± SD. One-way ANOVA assessed significance among three or more groups, and a Student's t-test was used for comparisons between two groups. Data analysis and image processing were conducted using Prism (GraphPad, Boston, MA, USA) and Photoshop CC (Adobe, San Jose, CA, USA). Statistical significance was defined as P < 0.05. All experiments were performed in at least three independent replicates. 

Results

LRRK2 Enhances A. fumigatus–Induced Inflammatory Response Through TLR2

We first investigated whether A. fumigatus can activate LRRK2. HCECs were stimulated with heat-inactivated A. fumigatus hyphae for 0, 1, 3, 6, 12, and 24 hours. Western blot analysis revealed that A. fumigatus significantly increased LRRK2 phosphorylation, with the highest level observed at 6 hours (Figs. 1A, 1B). TLR2, a member of the PRRs, plays a crucial role in the corneal innate immune response by directly detecting pathogenic fungi.^26–28 We then examined whether TLR2 is involved in A. fumigatus–induced LRRK2 activation. The results indicated that LRRK2 phosphorylation was significantly reduced in HCECs with TLR2 knockdown compared to the control group (Figs. 1C, 1D). Thus, our data suggest that A. fumigatus promotes LRRK2 activation through TLR2 in HCECs. 

Figure 1.

 

LRRK2 augments A. fumigatus–induced inflammatory response through TLR2. A. fumigatus hyphae (A. f; 1 × 10⁶ hyphal fragments/mL) were used to stimulate HCECs for 1, 3, 6, 12, and 24 hours. (A) Western blot was performed to detect the protein levels of p-LRRK2 (S935), LRRK2, and β-actin. (B) Quantification of p-LRRK2 protein expression shown in A. (C) HCECs were transfected with NC siRNA or TLR2 siRNAs (siTLR2-1 and siTLR2-2) for 24 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Protein levels of p-LRRK2 (S935), LRRK2, TLR2, and β-actin were detected by western blot. (D) Quantification of p-LRRK2 protein expression shown in C. HCECs were transfected with pcDNA3.1 (vector) or pcDNA3.1-LRRK2 (LRRK2) for 24 hours to overexpress LRRK2, followed by treatment with A. fumigatus hyphae for 12 hours. (E) Western blot was performed to detect the protein levels of p-LRRK2, LRRK2, and β-actin. (F) Quantification of the protein levels of p-LRRK2 and LRRK2 shown in E. (G) The mRNA expression of TNF-α, IL-6, and IL-1β was determined by qRT-PCR. (H) The protein expression of TNF-α, IL-6, and IL-1β in culture supernatants was detected by ELISA. Data are presented as the mean ± SD. ^#P > 0.05, *P < 0.05, **P < 0.01; n = 3.

Next, we aimed to clarify the role of LRRK2 in the inflammatory response induced by A. fumigatus. HCECs were transiently transfected with pcDNA3.1-LRRK2 to achieve LRRK2 overexpression, followed by treatment with A. fumigatus. The efficiency of LRRK2 overexpression was confirmed by western blot analysis (Figs. 1E, 1F). Consistent with our previous study,²⁵ A. fumigatus stimulation significantly increased the mRNA expression levels of inflammatory cytokines, including TNF-α, IL-6, and IL-1β, which were further elevated following LRRK2 overexpression (Fig. 1G). ELISA analysis also indicated that LRRK2 overexpression raised the protein levels of these inflammatory cytokines in HCECs stimulated with A. fumigatus (Fig. 1H). Therefore, these data suggest that LRRK2 overexpression promotes the inflammatory response in HCECs with A. fumigatus stimulation. 

Inhibition of LRRK2 Alleviates A. fumigatus–Induced Inflammatory Response

Considering that LRRK2 overexpression enhances inflammatory responses, our subsequent research focused on examining whether inhibiting LRRK2 activity could suppress the inflammation induced by A. fumigatus. HCECs were transfected with LRRK2-specific siRNAs (siLRRK2-1 and siLRRK2-2) or a negative control siRNA (siNC), followed by treatment with A. fumigatus. Western blot analysis confirmed that LRRK2 siRNAs successfully reduced LRRK2 protein levels (Fig. 2A). The quantitative reverse transcription polymerase chain reaction (qRT-PCR) results demonstrated that LRRK2 knockdown decreased the mRNA expression of inflammatory cytokines TNF-α, IL-6, and IL-1β in HCECs treated with A. fumigatus (Fig. 2B). ELISA analysis further showed that LRRK2 knockdown reversed the elevated levels of these inflammatory cytokines induced by A. fumigatus (Fig. 2C). Additionally, LRRK2-IN-1, a specific inhibitor of LRRK2, was utilized to inhibit LRRK2 activity (Fig. 2D).²⁹ The qRT-PCR and ELISA assays revealed that LRRK2-IN-1 significantly reduced the expression of TNF-α, IL-6, and IL-1β in HCECs stimulated with A. fumigatus (Figs. 2E, 2F). Also, we observed that LRRK2 siRNA or LRRK2-IN-1 alone did not affect the expression of inflammatory factors (Supplementary Fig. S2). These results suggest that inhibition of LRRK2 activity can effectively suppress the inflammatory response in HCECs during A. fumigatus stimulation. 

Figure 2.

 

LRRK2 inhibition alleviates A. fumigatus–induced inflammatory response. HCECs were transfected with NC siRNA or LRRK2 siRNAs (siLRRK2-1 and siLRRK2-2) for 24 hours to knock down LRRK2, followed by treatment with A. fumigatus hyphae (1 × 10⁶ hyphal fragments/mL) for 12 hours. (A) Western blot was performed to detect the protein levels of p-LRRK2, LRRK2, and β-actin. Quantification of the protein levels shown in A is provided in Supplementary Figure S1A. (B) The mRNA expression of TNF-α, IL-6, and IL-1β was determined by qRT-PCR. (C) The protein expression of TNF-α, IL-6, and IL-1β in culture supernatants was detected by ELISA. HCECs were pretreated with LRRK2-IN-1 (0, 1, and 2 µM) for 12 hours, followed by treatment with A. fumigatus hyphae for 12 hours. (D) Western blot was performed to detect the protein levels of p-LRRK2, LRRK2, and β-actin. Quantification of the protein levels shown in D is provided in Supplementary Figure S1B. (E) The mRNA expression of TNF-α, IL-6, and IL-1β was determined by qRT-PCR. (F) The protein expression of TNF-α, IL-6, and IL-1β in culture supernatants was detected by ELISA. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01; n = 3.

LRRK2 Regulates STING Phosphorylation Through Direct Interaction

To elucidate the mechanism by which LRRK2 regulates A. fumigatus keratitis, we performed a mass spectrometry–based proteomic analysis and identified STING as a novel interacting partner of LRRK2 (Fig. 3A). The GST–STING and His–LRRK2 were purified and the GST-pull down assay was used to confirm their direct interaction (Fig. 3B). The endogenous interaction between LRRK2 and STING was validated in HCECs using co-immunoprecipitation (co-IP) analysis (Fig. 3C). Additionally, to further verify the interaction, exogenous HA–LRRK2 and Myc–STING plasmids were transiently co-transfected into HEK293T cells, followed by Co-IP experiments. As demonstrated in Figures 3D and 3E, Myc–STING or HA–LRRK2 were detected among the proteins precipitated by anti-HA or anti-Myc antibodies, respectively. Next, we explored whether A. fumigatus stimulation affects the interaction between LRRK2 and STING. The results show that A. fumigatus treatment markedly enhanced this interaction (Fig. 3F). Numerous studies have reported that STING is activated through phosphorylation, subsequently mediating inflammatory responses induced by pathogenic microorganisms.^30,31 Ser366 of STING is the most important regulatory site involved in STING-mediated immune responses.^32,33 Our previous investigation also indicated that A. fumigatus stimulation activates the STING signaling pathway, thereby exacerbating fungal keratitis.³⁴ However, it remains unclear whether LRRK2 is responsible for STING activation during A. fumigatus treatment. Consistent with our previous findings, we observed that A. fumigatus increases STING phosphorylation in HCECs. On this basis, knockdown of LRRK2 reversed the upregulation of STING phosphorylation in HCECs induced by A. fumigatus stimulation (Fig. 3G), whereas LRRK2 overexpression led to an increase in STING phosphorylation (Fig. 3H). The reduced STING phosphorylation in HCECs with LRRK2 knockdown was restored upon reintroduction of LRRK2 (Fig. 3I). Moreover, the LRRK2-specific inhibitor LRRK2-IN-1 dramatically inhibited the phosphorylation of STING in HCECs treated with A. fumigatus (Figs. 3J, 3K). Additionally, the in vitro kinase assay confirmed that LRRK2 directly phosphorylates STING at Ser366 (Fig. 3L). Therefore, our data indicate that LRRK2 directly interacts with STING to promote its phosphorylation. 

Figure 3.

 

LRRK2 directly interacts with STING and regulates its phosphorylation. (A) HCECs transfected with either pcDNA3.1 (HA) or pcDNA3.1 LRRK2 (HA-LRRK2) were treated with A. fumigatus hyphae (1 × 10⁶ hyphal fragments/mL) for 6 hours. Following treatment, the cells were lysed and incubated with anti-HA beads. Then, the HA-IPed proteins were separated using SDS-PAGE gels and visualized by silver staining. (B) GST pull-down assay was conducted to validate the direct interaction between LRRK2 and STING. (C) Co-IP was performed to verify the endogenous interaction between LRRK2 and STING in HCECs. HA–LRRK2 and Myc–STING were co-transfected into HEK293T cells for 48 hours. (D) Co-IP assay was performed using anti-HA beads, and co-eluted Myc–STING was detected using anti-Myc antibody. (E) Co-IP assay was performed using anti-Myc beads, and co-eluted HA–LRRK2 was detected using anti-HA antibody. (F) A. fumigatus hyphae were used to stimulate HCECs for 12 hours, followed by pulldown with anti-LRRK2 antibody and immunoblotting with the antibodies indicated. (G) HCECs were transfected with NC siRNA or LRRK2 siRNAs (siLRRK2-1 and siLRRK2-2) for 24 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Western blot was performed to detect the protein levels of p-STING, STING, p-LRRK2, LRRK2, and β-actin. (H) HCECs were transfected with pcDNA3.1 (vector) or pcDNA3.1-LRRK2 (LRRK2) for 24 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Western blot was performed to detect the protein levels. (I) HCECs were transfected with NC siRNA, LRRK2 siRNA-2 (siLRRK2-2), or pcDNA3.1-LRRK2 (LRRK2) for 24 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Western blot was performed to detect the protein levels. (J) HCECs were pretreated with LRRK2-IN-1 (0, 1, and 2 µM) for 12 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Western blot was performed to detect the protein levels. (K) Quantification of the protein levels shown in J. (L) His–LRRK2 and GST–STING recombinant proteins were mixed in reaction buffer with or without phosphatase for 30 minutes. The protein levels of p-STING (Ser366), His–LRRK2, and GST–STING were detected using western blot. Quantification of protein levels in panels F to I is provided in Supplementary Figures S3A to S3D. Data are presented as the mean ± SD. **P < 0.01; n = 3.

LRRK2 Modulates STING Downstream Signaling Pathways

Activated STING plays a crucial role in mediating immune inflammatory responses by engaging various downstream signaling pathways, including TBK1, IRF3, and nuclear factor kappa B (NF-κB).³³ Given this, we further investigated the impact of LRRK2 on STING-associated signaling in HCECs with A. fumigatus stimulation. Western blot analysis revealed that LRRK2 overexpression led to increased protein levels of p-TBK1, p-IκBα, p-p65, and p-IRF3, indicating the activation of both IRF3 and NF-κB signaling pathways (Fig. 4A). Moreover, LRRK2 overexpression induced the nuclear translocation of p65, further supporting NF-κB activation (Fig. 4B and C). In addition, A. fumigatus treatment promotes the phosphorylation of TBK1, IκBα, p65, and IRF3. Knockdown of LRRK2 effectively abolished the increased phosphorylation of these proteins induced by A. fumigatus stimulation (Fig. 4D), whereas reconstituting LRRK2 reversed the reduction in phosphorylation observed with LRRK2 knockdown (Fig. 4E). Furthermore, our results revealed that the specific LRRK2 inhibitor LRRK2-IN-1 significantly suppressed the phosphorylation of TBK1, IκBα, p65, and IRF3 (Fig. 4F). We also found that LRRK2 siRNA or LRRK2-IN-1 alone did not impact the activation of STING-related pathways (Supplementary Fig. S5). To clarify whether STING is involved in LRRK2-mediated phosphorylation of these proteins, we first performed STING knockdown and subsequent reconstitution experiments in HCECs. Western blot analysis showed that LRRK2 failed to increase the levels of p-TBK1, p-IκBα, p-p65, and p-IRF3 in the absence of STING, but these effects were restored following STING reconstitution (Fig. 4G). Thus, these data indicate that LRRK2 activates STING downstream signaling pathways through its interaction with STING. 

Figure 4.

 

LRRK2 regulates STING downstream pathways. HCECs were transfected with pcDNA3.1 (vector) or pcDNA3.1-LRRK2 (LRRK2) for 24 hours, followed by treatment with A. fumigatus hyphae (1 × 10⁶ hyphal fragments/mL) for 6 hours. (A) Western blot was performed to detect the protein levels of p-TBK1, TBK1, p-IκBα, IκBα, p-p65, p65, p-IRF3, IRF3, p-LRRK2, LRRK2, and β-actin. (B) Western blot was performed to detect the protein levels of p65 in the cytoplasm and nucleus fractions. Lamin B1 and GAPDH were used to indicate cytoplasm and nucleus, respectively. (C) Immunofluorescence staining was performed to detect p65 nuclear translocation. (D) HCECs were transfected with NC siRNA or LRRK2 siRNAs (siLRRK2-1 and siLRRK2-2) for 24 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Western blot was performed to detect the protein levels. (E) HCECs were transfected with NC siRNA, LRRK2 siRNA-2 (siLRRK2-2), or pcDNA3.1-LRRK2 (LRRK2) for 24 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Western blot was performed to detect the protein levels. (F) HCECs were pretreated with LRRK2-IN-1 (0, 1, or 2 µM) for 12 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Western blot was performed to detect the protein levels. (G) HCECs were transfected with NC siRNA, LRRK2 siRNA-2 (siLRRK2-2), STING siRNA (siSTING), or pcDNA3.1-STING (STING) for 24 hours, followed by treatment with A. fumigatus hyphae for 6 hours. Western blot was performed to detect the protein levels. Quantification of protein levels shown in panels A to G is provided in Supplementary Figures S4A to S4G.

LRRK2 Regulates A. fumigatus–Induced Inflammatory Response Through Interacting With STING

Given that LRRK2 interacts with STING and regulates the activity of STING-related pathways, we investigated the involvement of STING in LRRK2-mediated A. fumigatus keratitis. We initially knocked down STING and then proceeded with STING reconstitution (Fig. 5A). The qRT-PCR and ELISA assays demonstrated that LRRK2 overexpression was unable to promote the expression of TNF-α, IL-6, and IL-1β in HCECs with STING knockdown, but reintroducing STING abrogated these effects (Figs. 5B, 5C). To further elucidate the effects of STING in A. fumigatus keratitis regulated by LRRK2, we applied a specific STING inhibitor, C176, to suppress STING activity (Fig. 5D). Results from qRT-PCR and ELISA demonstrated that administration of C176 reversed the elevated expression of TNF-α, IL-6, and IL-1β in HCECs with LRRK2 overexpression (Figs. 5E, 5F). Thus, our data suggest that LRRK2 regulates A. fumigatus–induced inflammatory response via STING in HCECs. 

Figure 5.

 

LRRK2 mediates A. fumigatus–induced inflammatory response through interacting with STING. HCECs were transfected with NC siRNA, LRRK2 siRNA-2 (siLRRK2-2), STING siRNA (siSTING), or pcDNA3.1-STING (STING) for 24 hours, followed by treatment with A. fumigatus hyphae (1 × 10⁶ hyphal fragments/mL) for 12 hours. (A) Western blot was performed to detect the protein levels of STING, p-LRRK2, LRRK2, and β-actin. (B) The mRNA expression of TNF-α, IL-6, and IL-1β was determined by qRT-PCR. (C) The protein expression of TNF-α, IL-6, and IL-1β in culture supernatants was detected by ELISA. HCECs were transfected with pcDNA3.1 (vector) or pcDNA3.1-LRRK2 (LRRK2) for 24 hours, followed by treatment with A. fumigatus hyphae for 12 hours. C176 (1 µM) was added to the medium for 24 hours before harvest. (D) Western blot was performed to detect the protein levels. (E) The mRNA expression of TNF-α, IL-6, and IL-1β was determined by qRT-PCR. (F) The protein expression of TNF-α, IL-6, and IL-1β in culture supernatants was detected by ELISA. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01; n = 3.

LRRK2 Knockdown Alleviates A. fumigatus Keratitis in Mouse

Mice are widely used as a model organism to study corneal infections globally.^35,36 In this study, we examined the role of LRRK2 in a mouse model of A. fumigatus keratitis. Mice were infected with live A. fumigatus hyphae for 0.5, 1, and 3 days, after which the corneas were harvested for investigation. Western blot analysis revealed a significant increase in the protein levels of p-LRRK2 and p-STING following A. fumigatus infection (Fig. 6A). The expression of p-LRRK2 and p-STING peaked at 1 day post-infection, leading us to conduct subsequent experiments with this time frame. To inhibit LRRK2 expression, LRRK2 siRNAs were subconjunctivally injected into mouse corneas 1 day before infection with A. fumigatus hyphae. The efficiency of LRRK2 knockdown is illustrated in Figure 6E. This knockdown resulted in reduced corneal ulceration and opacity, lower clinical scores, and diminished fungal burden compared to the control group (Figs. 6B–D). Consistent with observations in HCECs, A. fumigatus infection also increased the levels of p-STING in mouse corneas, an effect that was reversed by LRRK2 knockdown (Fig. 6E). Additionally, qRT-PCR and ELISA assays indicated that LRRK2 knockdown decreased the expression of inflammatory factors such as TNF-α, IL-6, and IL-1β (Figs. 6F, 6G). Thus, these results suggest that LRRK2 knockdown improves the inflammatory response induced by A. fumigatus infection in the mouse model. 

Figure 6.

 

Knockdown of LRRK2 inhibits A. fumigatus keratitis in mouse. (A) The mouse corneas were infected with live A. fumigatus hyphae (1 × 10⁸ hyphal fragments/mL, 5 µL) for 0.5, 1, and 3 days. The corneas were then excised for further study. Western blot was used to detect the protein levels of p-LRRK2 (S935), LRRK2, p-STING, STING, and β-actin. Quantification of the protein levels shown in A is provided in Supplementary Figure S6A. The mice were subconjunctivally injected with 5 µL NC siRNA (siNC, 10 µM) or 5 µL LRRK2 siRNAs (siLRRK2-1, siLRRK2-2; 10 µM) for 24 hours. The corneas were then infected with live A. fumigatus hyphae (5 µL) for 24 hours. Mouse corneas were harvested at 24 hours post-infection. (B) The severity of keratitis was assessed using slit-lamp examination. (C) Average clinical scores were calculated to assess the clinical manifestations. (D) Fungal plate counting was performed to assess fungal burden. (E) Protein expression of p-STING, STING, p-LRRK2, LRRK2, and β-actin in mouse corneas was detected by western blot. Quantification of the protein levels shown in E is provided in Supplementary Figure S6B. (F) The mRNA expression of TNF-α, IL-6, and IL-1β in mouse corneas was determined by qRT-PCR. (G) The protein expression of TNF-α, IL-6, and IL-1β in corneal homogenate was detected by ELISA. Data are presented as the mean ± SD. **P < 0.01; n = 6.

LRRK2 Inhibitor Ameliorates A. fumigatus Keratitis In Vivo

To further investigate the potential therapeutic effects of LRRK2 inhibitor in A. fumigatus keratitis, the specific LRRK2 inhibitor LRRK2-IN-1 was employed to inhibit LRRK2 activity in a mouse model. The slit-lamp images of mouse corneas revealed that LRRK2-IN-1 dramatically mitigated the corneal injury and inflammation resulting from A. fumigatus infection (Fig. 7A). Clinical scoring and fungal burden assessments also support these observations, indicating reduced disease severity in mouse corneas treated with LRRK2-IN-1 (Figs. 7B, 7C). Western blot analysis further confirmed that LRRK2-IN-1 effectively inhibited STING activity (Figs. 7D, 7E), consistent with the observations in HCECs. Additionally, qRT-PCR and ELISA results demonstrated a significant reduction in the levels of inflammatory cytokines, including TNF-α, IL-6, and IL-1β, following LRRK2 inhibition (Fig. 7F, 7G). Treatment with LRRK2 siRNA or LRRK2-IN-1 alone did not induce any observable changes in the cornea, nor did it affect STING pathway activation or inflammatory factor expression (Supplementary Fig. S7). Moreover, we found that LRRK2-IN-1 had no direct inhibitory effect on A. fumigatus within the concentration range used (Supplementary Fig. S8). Thus, our findings suggest that LRRK2 inhibitor exerts anti-inflammatory effects through STING in A. fumigatus-infected mouse corneas. 

Figure 7.

 

LRRK2 inhibitor ameliorates A. fumigatus keratitis in vivo. (A) The mouse corneas were infected with live A. fumigatus hyphae (1 × 10⁸ hyphal fragments/mL, 5 µL) for 24 hours. Then, 5 µL LRRK2-IN-1 (1 µg/µL or 2 µg/µL) was subconjunctivally injected 2 hours before A. fumigatus infection. Mouse corneas were harvested at 24 hours post-infection. The severity of keratitis was assessed using slit-lamp examination. (B) Average clinical scores were calculated to assess the clinical manifestations. (C) Fungal plate counting was performed to assess fungal burden. (D) Protein expression of p-LRRK2, LRRK2, p-STING, STING, and β-actin in mouse corneas was detected by western blot. (E) Quantification of the protein levels shown in D. (F) The mRNA expression of TNF-α, IL-6, and IL-1β in mouse corneas was determined by qRT-PCR. (G) The protein expression of TNF-α, IL-6, and IL-1β in corneal homogenate was detected by ELISA. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01; n = 6.

Discussion

The precise role of LRRK2 in immune regulation appears to vary based on specific cell types and the nature of the stimuli involved. In microglia and astrocytes, LRRK2 harboring the hyperactive PD mutations G2019S or R1441G leads to increased production of inflammatory cytokines and chemokines in response to various challenges.^14,37 Conversely, LRRK2 knockout (KO) or pharmacological inhibition tends to reduce the generation of inflammatory mediators, indicating that LRRK2 kinase activity positively regulates inflammation.^38,39 Interestingly, dendritic cells lacking LRRK2 show enhanced activation of the NF-κB pathway and increased production of inflammatory cytokines upon stimulation with lipopolysaccharide.⁴⁰ In a septic mouse model, the G2019S mutation provided protective effects against bacterial infection, although it was associated with higher mortality rates in pups infected with enterovirus-induced encephalitis.⁴¹ In this study, we found that LRRK2 was maintained in an inactive state in HCECs and normal mouse corneas, whereas A. fumigatus stimulation increased LRRK2 phosphorylation through TLR2. The active LRRK2 enhanced the production of inflammatory factors, including TNF-α, IL-6, and IL-1β, both in vitro and in vivo. Importantly, LRRK2 inhibition via siRNA knockdown or specific inhibitors alleviated the inflammatory response induced by A. fumigatus. Currently, the role of LRRK2 in fungal infections has not been reported. Our findings reveal the significant role of LRRK2 in fungal infections, contributing to the understanding of immune regulatory mechanisms in FK. 

The STING signaling pathway serves as a key regulatory factor in inflammatory responses to infection, cellular stress, and tissue damage.⁴² Upon activation, STING translocates from the endoplasmic reticulum to the Golgi apparatus, where it recruits and activates TBK1 and IκB kinase (IKK). These kinases subsequently phosphorylate and activate IRF3 and NF-κB, which promote the expression of type I interferons (IFNs) and other inflammatory cytokines.⁴³ Although the STING pathway mediates protective immune responses against pathogen infections, its aberrant activation due to self or pathogen DNA can contribute to autoimmune and inflammatory diseases.⁴⁴ Therefore, maintaining appropriate activation levels of the STING pathway is crucial. Our previous studies have demonstrated that A. fumigatus infection activates the STING signaling pathway, and inhibiting STING activity alleviates inflammation in FK.³⁴ However, the precise mechanisms governing STING activation in FK remain unclear. The phosphorylation of STING is critical for its activation and degradation processes. In addition to the classical phosphorylation by the second messenger cGAMP, activated STING can also be phosphorylated by ATG1, which serves as a negative feedback mechanism regulating STING activity.^45,46 Additionally, the estimated glomerular filtration rate (eGFR) induces the tyrosine phosphorylation of STING, facilitating its migration to endosomes where it activates IRF3.⁴⁷ The phosphatase PPMIA has also been shown to interact with STING, facilitating its dephosphorylation and inhibiting the antiviral signaling mediated by STING.⁴⁸ These insights reveal the intricate regulatory mechanisms of the STING pathway and the impact of different phosphorylation states on its function. In this study, we identified LRRK2 as a novel interaction partner of STING. We found that A. fumigatus treatment enhanced the interaction between LRRK2 and STING, leading to the phosphorylation and activation of STING. The phosphorylated STING subsequently exacerbated the severity of A. fumigatus keratitis. This research has uncovered a new mechanism by which STING is activated during the inflammatory response induced by A. fumigatus stimulation. 

Currently, numerous LRRK2 inhibitors with different chemical scaffolds and improved activity, selectivity, and pharmacokinetics have been developed, such as LRRK2-IN-1, GNE-7915, and CZC-25146.⁴⁹ The overactivation of LRRK2 has been demonstrated to have a strong association with PD; therefore, targeting LRRK2 is considered a promising therapeutic strategy for PD.⁵⁰ Clinical trials for DNL201, WXWH0226, NEU-723, and DNL151 in PD treatment are ongoing, with no severe adverse events reported.¹² Beyond their application in PD therapy, LRRK2 inhibitors have shown significant involvement in various cellular processes, including vesicular transport, immune responses, and autophagy. Vasoactive intestinal peptide regulates the actin cytoskeleton of Schlemm's canal endothelium by inducing Sp1-mediated LRRK2 expression, an effect that can be reversed by LRRK2-IN-1 both in vivo and in vitro.⁵¹ Pretreatment with the LRRK2 inhibitor LRRK2-IN-1 significantly reduces apoptotic speck protein containing a caspase recruitment domain (ASC) aggregation, caspase-1 activation, and IL-1β cleavage in peritoneal macrophages infected with Salmonella typhimurium.²⁰ Additionally, LRRK2 inhibitor CZC-25146 has been shown to promote autophagic responses, decrease polymer load, enhance normal alpha-1 antitrypsin (AAT) secretion, and subsequently reduce inflammatory cytokines in both cellular and liver injury mouse models.⁵² Here, we identified a novel function of the LRRK2 inhibitor LRRK2-IN-1 that significantly mitigates inflammatory response and corneal damage caused by A. fumigatus infection. We found that LRRK2-IN-1 inhibited the activation of STING, thereby suppressing the activity of its downstream signaling pathways. This inhibition ultimately led to a reduction in the inflammatory response. Our findings indicate that targeting LRRK2 may represent a promising therapeutic strategy for FK. 

In our in vivo experiments, we observed that inhibiting LRRK2 activity led to a reduction in both the inflammatory response and fungal load. We have demonstrated that LRRK2 modulates the inflammatory response through the STING signaling pathway in HCECs. The reduction in fungal load may be attributed to enhanced phagocytosis and increased spore-killing activity of immune cells, such as macrophages or neutrophils. However, the exact mechanisms by which LRRK2 influences immune cell-mediated fungal killing remain to be further elucidated. Additionally, our current research primarily focuses on the role of LRRK2 at the cellular level and in mouse models. The efficacy of LRRK2 inhibitors in improving FK requires further validation in clinical settings. 

In conclusion (Fig. 8), our findings showed that A. fumigatus stimulation activates LRRK2, which subsequently phosphorylates and activates its interaction partner STING. Activated STING further triggers downstream signaling pathways involving IRF3 and NF-κB, ultimately promoting the inflammatory response induced by A. fumigatus treatment. Importantly, LRRK2 inhibitors alleviated A. fumigatus keratitis by inhibiting STING activation. Our study has elucidated the mechanism by which the LRRK2/STING pathway regulates the progression of FK, highlighting that targeting LRRK2 is a potential strategy for treating FK. 

Figure 8.

 

Working model of how LRRK2/STING mediates FK. A. fumigatus stimulation activated LRRK2 through TLR2. Once activated, phosphorylated LRRK2 interacted with STING, leading to the phosphorylation and activation of STING via its kinase activity. This activated STING subsequently triggered downstream signaling pathways involving IRF3 and NF-κB, ultimately promoting the inflammatory response induced by A. fumigatus treatment. Furthermore, LRRK2 inhibitor mitigated A. fumigatus keratitis by inhibiting STING-related signaling pathways.

Acknowledgments

The authors think Fu-Shin Yu, PhD, at Wayne State University for generously providing the HCECs for our study. 

Supported by grants from the National Natural Science Foundation of China (82201155, 82401220) and the Natural Science Foundation of Shandong Province (ZR2022QH326). 

Disclosure: F. Han, None; L. Wang, None; J. Wu, None; L. Shen, None; Y. Li, None; H. Guo, None; J. Li, None