Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of LRRK2 Ameliorates A spergillus fumigatus Keratitis by Regulating STING Signaling Pathways

doi: 10.1167/iovs.66.2.13

Figure Lengend Snippet: LRRK2 augments A. fumigatus –induced inflammatory response through TLR2. A. fumigatus hyphae (A. f; 1 × 10 6 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.

Article Snippet: A. fumigatus (strain 93024) was sourced from the China Center for Type Culture Collection (CCTCC, Beijing, China).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of LRRK2 Ameliorates A spergillus fumigatus Keratitis by Regulating STING Signaling Pathways

doi: 10.1167/iovs.66.2.13

Figure Lengend Snippet: 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 × 10 6 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 A. ( 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 B. ( 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.

Article Snippet: A. fumigatus (strain 93024) was sourced from the China Center for Type Culture Collection (CCTCC, Beijing, China).

Techniques: Inhibition, Transfection, Knockdown, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of LRRK2 Ameliorates A spergillus fumigatus Keratitis by Regulating STING Signaling Pathways

doi: 10.1167/iovs.66.2.13

Figure Lengend Snippet: 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 × 10 6 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 A to D. Data are presented as the mean ± SD. ** P < 0.01; n = 3.

Article Snippet: A. fumigatus (strain 93024) was sourced from the China Center for Type Culture Collection (CCTCC, Beijing, China).

Techniques: Phospho-proteomics, Transfection, Incubation, SDS Page, Silver Staining, Pull Down Assay, Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Recombinant

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of LRRK2 Ameliorates A spergillus fumigatus Keratitis by Regulating STING Signaling Pathways

doi: 10.1167/iovs.66.2.13

Figure Lengend Snippet: 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 × 10 6 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 A to G.

Article Snippet: A. fumigatus (strain 93024) was sourced from the China Center for Type Culture Collection (CCTCC, Beijing, China).

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunofluorescence, Staining, Translocation Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of LRRK2 Ameliorates A spergillus fumigatus Keratitis by Regulating STING Signaling Pathways

doi: 10.1167/iovs.66.2.13

Figure Lengend Snippet: 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 × 10 6 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.

Article Snippet: A. fumigatus (strain 93024) was sourced from the China Center for Type Culture Collection (CCTCC, Beijing, China).

Techniques: Transfection, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of LRRK2 Ameliorates A spergillus fumigatus Keratitis by Regulating STING Signaling Pathways

doi: 10.1167/iovs.66.2.13

Figure Lengend Snippet: Knockdown of LRRK2 inhibits A. fumigatus keratitis in mouse. ( A ) The mouse corneas were infected with live A. fumigatus hyphae (1 × 10 8 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 A. 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 B. ( 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.

Article Snippet: A. fumigatus (strain 93024) was sourced from the China Center for Type Culture Collection (CCTCC, Beijing, China).

Techniques: Knockdown, Infection, Western Blot, Injection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of LRRK2 Ameliorates A spergillus fumigatus Keratitis by Regulating STING Signaling Pathways

doi: 10.1167/iovs.66.2.13

Figure Lengend Snippet: LRRK2 inhibitor ameliorates A. fumigatus keratitis in vivo. ( A ) The mouse corneas were infected with live A. fumigatus hyphae (1 × 10 8 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.

Article Snippet: A. fumigatus (strain 93024) was sourced from the China Center for Type Culture Collection (CCTCC, Beijing, China).

Techniques: In Vivo, Infection, Injection, Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of LRRK2 Ameliorates A spergillus fumigatus Keratitis by Regulating STING Signaling Pathways

doi: 10.1167/iovs.66.2.13

Figure Lengend Snippet: 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.

Article Snippet: A. fumigatus (strain 93024) was sourced from the China Center for Type Culture Collection (CCTCC, Beijing, China).

Techniques: Phospho-proteomics, Activation Assay, Activity Assay, Protein-Protein interactions