Flavopiridol Protects against Fungal Keratitis due to Aspergillus fumigatus by Alleviating Inflammation through the Promotion of Autophagy

Abstract

Fungal keratitis is a serious infectious keratopathy related to fungal virulence and excessive inflammatory responses. Autophagy exhibits a potent ability to resolve inflammation during fungal infection. This study aimed to investigate the protective function of flavopiridol in Aspergillus fumigatus keratitis and explore its effects on autophagy. In our study, the corneas of the fungal keratitis mouse model were treated with 5 μM flavopiridol. In vitro, RAW 264.7 cells were pretreated with 200 nM flavopiridol before fungal stimulation. A. fumigatus was incubated with flavopiridol, and the antifungal activity of flavopiridol was detected. Our results indicated that flavopiridol treatment notably reduced clinical scores as well as cytokines expression of infected corneas. In infected RAW 264.7 cells, flavopiridol treatment inhibited IL-1β, IL-6, and TNF-α expression but promoted IL-10 expression. Transmission electron microscopy (TEM) images showed that more autolysosomes were present in infected corneas and RAW 264.7 cells after flavopiridol treatment. Flavopiridol treatment notably upregulated the protein expression of LC3, Beclin-1, and Atg-7. 3-Methyladenine (3-MA, an inhibitor of autophagy) pretreatment counteracted the cytokine regulation induced by flavopiridol. Moreover, flavopiridol promoted the phagocytosis of RAW 264.7 cells. Flavopiridol also exhibited antifungal activity by restricting fungal growth and limiting fungal biofilm formation and conidial adhesion. In conclusion, flavopiridol significantly alleviated the inflammation of fungal keratitis by activating autophagy. In addition, flavopiridol promoted the phagocytosis of RAW 264.7 cells and exhibited antifungal function, indicating the potential therapeutic role of flavopiridol in fungal keratitis.

The Pharmacological Implications of Flavopiridol: An Updated Overview

“…Chang and his colleagues reported the abrogation of inflammatory mediators in FP alone or in combination with PLGA (poly (lactic-co-glycolic acid)) nanoparticle-treated astrocytes through significant inhibition of pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNF-α) and induction of an anti-inflammatory cytokine (i.e., IL-10) [104,113]. Similar results were obtained in a fungal keratitis mouse model, wherein FP inhibited the inflammation induced in fungal keratitis through the production of the IL-1β, IL-6, and TNF-α cytokines while promoting the production of the IL-10 cytokine [29]. All the evidence indicated herein points towards FP's ability to inhibit inflammation through a multitude of mechanisms, as discussed above, making it a potential anti-inflammatory agent for future clinical research.…”

“…It has been observed that it abrogates the replication of different viruses, such as human immunodeficiency virus (HIV), herpes simplex virus (HSV1 and 2), human cytomegalovirus (HCMV), human adenovirus (hAdV5 and hAdV53), and influenza virus A (HINI, H3N2, H7N9) by suppression of mRNA transcription through P-TEFb/CDK9 complex inhibition [26][27][28]116,117]. Similarly, it inhibits the growth of different parasites, including Leishmania mexicana, Toxoplasma gondii, and Plasmodium falciparum [29,118]. Interestingly, it has been noted that FP inhibits fungal growth, biofilm formation, and the adhesion ability of Aspergillus fumigatus, decreases inflammation, and induces autophagy [29].…”

“…Similarly, it inhibits the growth of different parasites, including Leishmania mexicana, Toxoplasma gondii, and Plasmodium falciparum [29,118]. Interestingly, it has been noted that FP inhibits fungal growth, biofilm formation, and the adhesion ability of Aspergillus fumigatus, decreases inflammation, and induces autophagy [29]. Aside from these, it has several other pharmacological benefits, described in Table 1, and the underlying molecular mechanisms have been proposed.…”

“…Flavopiridol has been extensively explored for its pharmacological therapeutic implications over the last few decades. Flavopiridol modulates several signaling pathways associated with tumor proliferation and metastasis, viral replication, fungal infection, and inflammation-associated diseases [26][27][28][29][30]. Even though such bioactive semi-synthetic compounds are available, their use in treating cancer and viral, fungal, and inflammationassociated diseases is yet to be determined.…”

Progranulin Protects against Aspergillus fumigatus Keratitis by Attenuating the Inflammatory Response through Enhancing Autophagy

“…12 The fusion of autophagic vesicles with lysosomes and the formation of autophagic lysosomes were specifically inhibited by 3-MA. 28 In the current study, RAW 264.7 cells and mice were treated with the autophagy inhibitor 3-MA and then given PGRN treatment. The findings of our study demonstrated that treatment with 3-MA effectively counteracted the regulatory role of PGRN in cytokine expression and the amelioration of corneal injury during fungal infection.…”