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. 2024 Jul 1;65(8):4.
doi: 10.1167/iovs.65.8.4.

The Role of LC3-Associated Phagocytosis Inhibits the Inflammatory Response in Aspergillus fumigatus Keratitis

Junjie Luan  1 , Ziyue Zhang  1   2 , Qian Wang  1 , Cui Li  1 , Hao Zhang  1 , Yingxue Zhang  3 , Xudong Peng  1 , Guiqiu Zhao  1 , Jing Lin  1
Affiliations

The Role of LC3-Associated Phagocytosis Inhibits the Inflammatory Response in Aspergillus fumigatus Keratitis

Junjie Luan et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: The purpose of this study was to investigate the role and mechanism of microtubule-associated protein light chain-3 (LC3)-associated phagocytosis (LAP) in the immune response to Aspergillus fumigatus (A. fumigatus) keratitis.

Methods: The formation of single-membrane phagosomes was visualized in the corneas of healthy or A. fumigatus-infected humans and C57BL/6 mice using transmission electron microscopy (TEM). Rubicon siRNA (si-Rubicon) was used to block Rubicon expression. RAW 264.7 cells or mice corneas were infected with A. fumigatus with or without pretreatment of si-Rubicon and scrambled siRNA. RAW 264.7 cells were pretreated with Dectin-1 antibody or Dectin-1 overexpressed plasmid and then stimulated with A. fumigatus. Flow cytometry was used to label macrophages in normal and infected corneas of mice. In mice with A. fumigatus keratitis, the severity of the disease was assessed using clinical scores. We used lentiviral technology to transfer GV348-Ubi-GFP-LC3-II-SV40-Puro Lentivirus into the mouse cornea. The GFP-LC3 fusion protein was visualized in corneal slices using a fluorescence microscope. We detected the mRNA and protein expressions of the inflammatory factors IL-6, IL-1β, and IL-10 using real-time PCR (RT-PCR) and ELISA. We detected the expression of LAP-related proteins Rubicon, ATG-7, Beclin-1, and LC3-II using Western blot or immunofluorescence.

Results: Accumulation of single-membrane phagosomes within macrophages was observed in the corneas of patients and mice with A. fumigatus keratitis using TEM. Flow cytometry (FCM) analysis results show that the number of macrophages in the cornea of mice significantly increases after infection with A. fumigatus. LAP-related proteins were significantly elevated in the corneas of mice and RAW 264.7 cells after infection with A. fumigatus. The si-Rubicon treatment elevated the clinical score of mice. In A. fumigatus keratitis mice, the si-Rubicon treated group showed significantly higher expression of IL-6 and IL-1β and lower expression of IL-10 and LC3-II compared to the control group. In RAW 264.7 cells, treatment with the Dectin-1 overexpressed plasmid upregulated the expression of LAP-related proteins, a process that was significantly inhibited by the Dectin-1 antibody.

Conclusions: LAP participates in the anti-inflammatory immune process of fungal keratitis (FK) and exerts an anti-inflammatory effect. LAP is regulated through the Dectin-1 signaling pathway in A. fumigatus keratitis.

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Conflict of interest statement

Disclosure: J. Luan, None; Z. Zhang, None; Q. Wang, None; C. Li, None; H. Zhang, None; Y. Zhang, None; X. Peng, None; G. Zhao, None; J. Lin, None

Figures

Figure 1.
Figure 1.
Single-membrane phagosomes formation in patients and mice with A. fumigatus keratitis. TEM images showed the corneal stroma in both healthy humans (A) and mice (D). Additionally, TEM images illustrated macrophages within the corneal stroma of humans infected with A. fumigatus (B, C). Similar images were captured of the corneal stroma in mice at 3 days pi. Black arrows highlight single-membrane phagosomes in the macrophages (B, C, E, F). Slit lamp images of mice corneas (G) were obtained at 1, 3, and 5 days pi., showing a significant increase in clinical scores at 3 days pi (H). Normal and A. fumigatus-infected corneas were collected for FCM analysis. Gating on single cells, CD45+ cells were immune cells, and CD45+ F4/80+ cells were macrophages. Representative flow cytometric plots (I, J) and data showed the number of macrophages in corneas (K). Clinical scores were analyzed using the Mann–Whitney U test. ***P < 0.001.
Figure 2.
Figure 2.
Expression of LAP-related protein in A. fumigatus keratitis mouse models. β-actin is a housekeeping protein whose stable expression is relatively constant in each group and is used to an internal control for protein expression normalization in Western blotting. Rubicon (A, B), ATG-7 (C, D), Beclin-1 (E, F), and LC3-II (G, H) protein levels were significantly increased at 1, 3, and 5 days after infection (n = 6 mice/group). Corneas of mice were transfected with GV348-Ubi-GFP-LC3-II-SV40-Puro Lentivirus. The GFP-LC3 fusion protein (green) was visualized in corneal slices at a magnification of 400× using a fluorescence microscope on the third day pi (n = 6 mice/group). The expression of LC3 was significantly higher in mice corneas at 3 days pi (I, J). All data were mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3.
Figure 3.
The effect of LAP on the inflammatory response. RAW 264.7 cells were pretreated with si-Rubicon or scrambled siRNA for 24 hours, and then incubated with A. fumigatus for 8 or 24 hours. RT-PCR results showed that the expression level of Rubicon mRNA was significantly reduced in RAW 264.7 cells treated with si-Rubicon (A). Corneas of mice were pretreated with si-Rubicon or scrambled siRNA for 2 days and then stimulated with A. fumigatus. Western blot results and grayscale analysis demonstrated that the protein expression levels of Rubicon (B, C) and LC3-II (D, E) were significantly reduced in the si-Rubicon treatment groups. Clinical scores were significantly higher in the si-Rubicon treatment groups (F, G). The mRNA and protein levels of IL-6 (H, I, J, K), IL-1β (I, J, K, L), and IL-10 (J, K, L, M) in the corneas of mice after si-Rubicon treatment at 3 days pi. Clinical scores were analyzed using the Mann–Whitney U test. All data were mean ± SEM (ns = no significance; *P < 0.05; **P < 0.01; ***P < 0.001.)
Figure 4.
Figure 4.
Dectin-1 regulates the expression of Lap-related proteins in A. fumigatus keratitis. RAW 264.7 cells were incubated with A. fumigatus hyphae for 12 and 24 hours. Western blot results showed that the protein expression levels of Rubicon (A, B), ATG-7 (C, D), Beclin-1 (E, F), and LC3-II (G, H) were significantly increased after stimulation by A. fumigatus. Immunofluorescence images showed the expression of Rubicon (I), ATG-7 (K), and Beclin-1 (M) in A. fumigatus–infected RAW 264.7 cells. Proportion of Rubicon (J), ATG-7 (L), and Beclin-1 (N) positive cells. Green = Rubicon, ATG-7, Beclin-1 (FITC); blue = nucleus (DAPI). RAW 264.7 cells were treated with Dectin-1 polyclonal antibody or Dectin-1 overexpressed plasmids, and then incubated with A. fumigatus hyphae for 24 hours. The protein levels of Rubicon (O, P. Q, R), ATG-7 (P, Q, R, S), Beclin-1 (Q, R, S, T), and LC3-II (U, V, W, X) were significantly decreased in the Dectin-1 antibody pretreated groups. Conversely, the protein levels of LC3-II (V, W, X, Y) and Rubicon (W, X, Y, Z) were significantly increased in the groups pretreated with the Dectin-1 overexpression plasmid. All data were mean ± SEM (ns = no significance; *P < 0.05; **P < 0.01; ***P < 0.001.) Magnification, 400 × (I, K, M).
Figure 4.
Figure 4.
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