ATF4 Involvement in TLR4 and LOX-1-Induced Host Inflammatory Response to Aspergillus fumigatus Keratitis

Abstract

Purpose Activating transcription factor 4 (ATF4) is induced by various stressors. Here, we investigated the expression of ATF4 in the host inflammatory response to Aspergillus fumigatus (A. fumigatus) keratitis. Methods A. fumigatus keratitis mouse models developed by intrastromal injection as well as corneal epithelium scratching were examined daily with a slit lamp microscope for corneal opacification and ulceration. Subsequent in vitro experimentation was carried out in human corneal epithelial cells (HCECs) as well as THP-1 macrophages infected with A. fumigatus. Inhibitors, including CLI-095, Poly (I), SCH772984, and SP600125, were used to assess the role of proteins like toll-like receptor 4 (TLR4), lectin-type oxidized LDL receptor 1 (LOX-1), extracellular signal-regulated kinases (ERK1/2), and c-Jun N-terminal kinase (JNK) in ATF4 expression as a response to A. fumigatus infection. This assessment was made in both mouse models and HCECs using western blot. Results Compared to the controls, ATF4 was increased in corneas from two kinds of A. fumigatus keratitis models at 3 days after infection. ATF4 expression was upregulated with A. fumigatus conidia both in HCECs and THP-1 macrophages 16 hours after stimulation. Furthermore, ATF4 expression in response to A. fumigatus infection was shown to be dependent on TLR4 and LOX-1 expression, and ERK1/2 and JNK contributed to the expression of ATF4 in response to A. fumigatus. Conclusion Our results clearly indicate that ATF4 was involved in the host antifungal immune response to A. fumigatus keratitis; expression was found to be dependent on TLR4, LOX-1 expression, and MAPKs pathway.

Role of Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in inflammation and pathogen-associated interactions

“…Furthermore, in macrophages and neutrophils, it was shown that HMGB1 upregulates LOX-1 through the DNA-binding domain BoxB [ 111 ]. Another inflammatory factor involved in the host immune response is activating transcription factor 4 (ATF4), which was shown to be dependent on LOX-1, ERK1/2, TLR4, and the JNK pathway in fungal keratitis [ 141 ]. It was also observed that inhibiting LOX-1 leads to a decrease in protein levels of adhesion factors such as VCAM-1, ICAM-1, P-selectin, E-selectin, and LFA-1.…”

Experimental Models for Fungal Keratitis: An Overview of Principles and Protocols

“…Then, a 33G Hamilton syringe is inserted into the tunnel to deliver a 1-2 µL inoculum directly into the underlying stroma. Regardless of fungal species, 10 5 total conidia is commonly used inoculum, although this can vary by a log-fold either way [1,32,[47][48][49][50][51][52].…”

“…For the inoculum, 5 µL of a conidial suspension (10 8 /mL for 2 groups using A. fumigatus ) or a non-specific smear of fungi can be applied to the debrided cornea, followed by contact lens placement. The contact lens must be secured in place by sewing the eyelids shut [ 31 , 51 , 53 , 54 , 55 ]. As an example of one such study, Huang et al performed an intrastromal injection of 10 8 /mL F. solani as described above and then placed a contact lens engineered to have antimicrobial properties onto the eye [ 56 ].…”