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Published on: July 9, 2014
PFKFB3 alleviates the advancement of Fusarium solani keratitis by attenuating macrophage inflammation
Yani Zhang1, Yanqing Zhang1, Hanfeng Tang1
1Department of Ophthalmology, Fujian Medical University Union Hospital, Fuzhou, China.
Objective:
To investigate the anti-inflammatory effect of glycolysis rate-limiting enzyme 6-phosphofructose-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3) in fungal keratitis (FK) infected by Fusarium solani (F. solani).
Methods:
We identified the up-regulation of PFKFB3 in fungal keratitis via western blot, quantitative real-time polymerase chain reaction (RT-PCR), and immunofluorescence staining. Subsequently, elucidated the augmentation of glycolytic flux in cornea and bone marrow-derived macrophages (BMDM) following F. solani invasion by RT-PCR, cellular energy metabolism analyzer, and lactate content assay. After that, we reduced PFKFB3 expression utilizing small interfering RNA (siRNA) in vitro and adeno-associated virus (AAV) in vivo and also assessed the expression levels of inflammatory factors. The severity of corneal infection following PFKFB3 depletion was checked by slit-lamp microscopy, corneal OCT, and H&E staining. Ultimately, we assessed the phosphorylation status of the PI3K/AKT/NF-κB p65 signaling pathway following PFKFB3 suppression via western blot and immunofluorescence staining.
Results:
PFKFB3 was highly triggered in F. solani-infected corneas and BMDM compared to normal tissue. Besides, infection with F. solani promotes the increase of inflammatory mediators and glycolytic flux in the cornea and BMDM. Whereas inflammation in BMDM and the degree of fungal keratitis lesions worsen by suppressing PFKFB3 expression, which increased corneal ulcer infiltration, elevated clinical scores, enhanced corneal thickness, and upregulation of inflammatory signals could be demonstrated. Furthermore, we found that F. solani infection can activate the phosphorylation of PI3K/AKT/NF-κB p65 at low PFKFB3 expression levels.
Conclusions:
In F. solani-infected corneas and BMDM, the glycolysis rate-limiting enzyme PFKFB3 was markedly upregulated. After infection, moderate PFKFB3 activation effectively mitigates inflammation and the progression of fungal keratitis. Moreover, activated PFKFB3 may rely on the PI3K/AKT/NF-κB p65 signaling pathway to safeguard the cornea from further damage due to inflammation.
Insights
The enzyme 6-phosphofructose-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3) is upregulated during Fusarium solani fungal keratitis. Moderate PFKFB3 activation mitigates inflammation and disease progression, potentially via the PI3K/AKT/NF-κB pathway.
Area of Science:
- Ophthalmology
- Immunology
- Molecular Biology
Background:
- Fungal keratitis (FK) is a serious eye infection.
- Fusarium solani (F. solani) is a common cause of FK.
- The role of metabolic enzymes in FK pathogenesis is not fully understood.
Purpose of the Study:
- To investigate the anti-inflammatory effect of 6-phosphofructose-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3) in F. solani-induced fungal keratitis.
- To elucidate the underlying molecular mechanisms.
Main Methods:
- Western blot, RT-PCR, and immunofluorescence staining were used to detect PFKFB3 expression.
- Glycolytic flux and inflammatory mediators were assessed in corneal and bone marrow-derived macrophage (BMDM) models.
- PFKFB3 was suppressed using siRNA in vitro and AAV in vivo.
- Corneal infection severity was evaluated using clinical scores, OCT, and H&E staining.
- PI3K/AKT/NF-κB p65 signaling pathway activation was analyzed.
Main Results:
- PFKFB3 was significantly upregulated in F. solani-infected corneas and BMDM.
- F. solani infection increased glycolytic flux and inflammatory mediators.
- Suppression of PFKFB3 exacerbated inflammation and fungal keratitis severity.
- Activated PFKFB3 mitigated inflammation and disease progression.
- F. solani infection activated PI3K/AKT/NF-κB p65 signaling.
Conclusions:
- PFKFB3 is upregulated in fungal keratitis and plays a protective role.
- Moderate PFKFB3 activation mitigates inflammation and fungal keratitis progression.
- The PI3K/AKT/NF-κB signaling pathway is involved in PFKFB3-mediated protection.
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