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

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.


Introduction
As severe infectious corneal diseases are mostly caused by Fusarium solani and Aspergillus fumigatus, fungal keratitis is likely to damage visual acuity and cause blindness [1][2][3]. Separate from Acanthamoeba keratitis, the typical clinical features of fungal keratitis are difficult to ascertain in the early stage of infection, leading to common misdiagnoses and delayed treatment [4,5]. Although there are new therapies used in clinical application, fungal keratitis remains a challenge to ophthalmologists due to a lack of effective drugs after late initiation of treatment secondary to delayed diagnosis [6,7].
ATF4, also known as tax-responsive enhancer element B67, is a protein encoded by the ATF4 gene in human. ATF4 expression is induced by ER as well as oxidative stress and is upregulated by eukaryotic initiation factor 2 phosphorylation in the protein kinase R-like ER kinase pathway. ATF4 has also been shown to be involved in the TLR-mediated innate immune response [14,15]. However, the expression of ATF4 in fungal keratitis has not been previously studied. To this end, we executed in vivo and in vitro experiments in mouse models, human corneal epithelial cells (HCECs), and THP-1 macrophages infected with A. fumigatus to fill this knowledge gap. We were able to confirm that ATF4 was involved in host antifungal immune response in A. fumigatus keratitis and was dependent on TLR4, LOX-1 expression, and MAPKs pathway.

Preparation of A. fumigatus.
A. fumigatus strain 3.0772 was purchased from the China General Microbiological Culture Collection Center (Beijing, China) and was cultured for 3-4 days on Sabouraud agar. Suspensions of fresh conidia scraped from the medium surface were quantified using a haemocytometer and adjusted to a final concentration of 5 × 10 4 conidia/µL in phosphate-buffered saline (PBS).

Keratitis Mouse Model Developed by Intrastromal
Injection. Eight-week-old C57BL/6 female mice were purchased from the Changzhou Cavens Laboratory (Jiangsu, China). Experimental mice were treated in accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research by the Association for Research in Vision and Ophthalmology (ARVO). Mice were anaesthetized with 8% chloral hydrate, and then one eye was randomly selected from each mouse. A. fumigatus conidia (2 µL, concentration 0.5 × 10 5 µL) were released into the corneal stroma of the mouse [16], and the mice were subsequently examined by a slit lamp microscope for corneal opacification and ulceration. Corneas were harvested for use in western blot assay at 1/2, 1, 2, 3, 5, 7, 10, and 14 days after infection.

Keratitis Model Developed by Corneal Epithelium Scratch.
One eye was randomly selected from one mouse. e central corneas were scratched (making a three 1 mm incision) using a sterile 25 5/8 gauge needle, and then the scratches were covered with A. fumigatus conidia (5 µL, concentration 1 × 10 8 µL). e eyelid was then sutured with a soft contact lens to cover the corneal surface [11]. Mice were examined by a slit lamp microscope, and corneas were harvested for western blot assay at 1, 3, and 5 days after infection.

2.5.
Culture of THP-1 Macrophages. THP-1 macrophages were purchased from the China Center for Type Culture Collection (Wuhan, China) and differentiated with 100 nM of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, USA) for 48 hours then allowed to recover for 24 hours prior to infection. Macrophages were cultured in RPMI-1640 medium at a density of 1 × 10 6 mL and then were treated in 6-well plates with 100 ng/mL LPS, 5 µg/mL β-1, 3-glucan, 1 ng/mL TGF-β, 10 ng/mL IL-1β, and finally A. fumigatus conidia at a MOI of 1 for 8 and 16 hours. eses samples were then analyzed with western blot assay.

Western Blot Assay.
e western blot assay protocol used for analyzing corneas and cell samples has been described in previous publications [12,16]. Membranes were incubated accordingly with anti-ATF4 (ProteinTech, USA) and anti-β-actin (CST, USA). e HRP-tagged secondary antibodies used were purchased from CST. e Fusion Solo system (VilberLourmat, France) developed blots with ECL substrate; additionally, histograms were used to quantify and represent chemiluminescence.

Statistical
Analysis. An unpaired two-tailed Student's ttest was used to determine the statistical significance of western blot data and was represented as mean ± SD and was analyzed using GraphPad 5.0 software. Data were considered significant at P ≤ 0.05.

ATF4 Was Increased in Mouse Corneas Infected by
Intrastromal Injection. Significant corneal opacity developed in C57BL/6 mouse models at 1 day after infection via intrastromal injection. is opacity persisted up to 14 days (Figure 1(a)) but corneal inflammation gradually improved with increased neovascularization. Compared with controls, ATF4 protein was degraded in infected mouse corneas at 1 day after infection (P < 0.05), then elevated at 3 days, and persisted up to 7 days (P < 0.05). As inflammation subsided, the expression of ATF4 returned to normal 10 days after infection (P > 0.05) (Figure 1(b)).

Discussion
Our results demonstrate that ATF4 was involved in the host antifungal immune response to A. fumigatus keratitis. is involvement was dependent on expression of TLR4, LOX-1, ERK1/2, and the JNK pathway. Although the role of ATF4 in fungal keratitis is still not well understood, many studies have provided support to its role in inflammation. For example, Woo et al. [22] demonstrated that ATF4 mediated the upregulation of rottlerin-induced COX-2 via the p38 mitogen-activated protein kinase (MAPK) pathway. en, Hayashi et al. [23] examined ATF4 and found it suppressed activation of T cells by inhibiting L-type amino acid transporter 1 and increasing homeobox B9 expression induced by amino acid deprivation. Furthermore, Yang et al. [24] showed mediation by the EIF2AK4/GCN2-EIF2S1/ eIF2α-ATF4 pathway in Pseudomonas aeruginosa-induced autophagy-enhanced bacterium fitness in human airways. Taken together, these observations are consistent with our results that ATF4 was involved in host immune response to an infectious disease.
Importantly, ATF4 levels were increased in corneas, compared to controls in mouse A. fumigatus keratitis models developed by intrastromal injection or corneal epithelium scratch 3 days after infection. Being the most commonly used mouse models for fungal keratitis research, both models showed similar ATF4 expression trends. As inflammation subsided, the expression of ATF4 returned to normal. Our findings indicate that as an ER stress-associated transcription factor, ATF4, is involved in the host immune response especially in the most serious stages of A. fumigatus keratitis.
Additionally, we confirmed that ATF4 expression was upregulated with the addition of A. fumigatus conidia both in HCECs and THP-1 macrophages at 16 hours after stimulation. Fungal keratitis is often caused by ocular trauma resulting from eye irritation by vegetative matter [1,25]. From an anatomical point of view, the epithelium is  one of the earliest corneal structures to come in contact with pathogenic fungi. For this reason, human corneal epithelial cells have been shown to be involved in antifungal immunity by releasing antimicrobial peptides, inflammatory cytokines, and chemokines [26][27][28]. Neutrophils are then recruited to the cornea to fight against fungal infection [29][30][31].Our findings additionally indicate that ATF4 was involved in fungal keratitis both with regards to the corneal epithelium and immunocytes. ATF4 has been shown to be induced by multiple types of stimuli. For example, Sasaki et al. [17] identified ATF4 as a mediator of IL-6 expression induced by LPS in embryonic fibroblast cells of p32 deficient mouse. Hara et al. [32] identified that ATF4 mediated IL-23 p19 mRNA expression  induced by LPS in a JNK-dependent manner, which could be inhibited by apomorphine. Our results are consistent with reports that ATF4 expression was upregulated with LPS stimulation in THP-1 macrophages, but not in HCECs.
TGF-β is a Treg-specific cytokine [33,34], and IL-1β is a 1 proinflammatory cytokine [35,36] previously demonstrated to be correlated with ATF4 expression. For example, Liu et al. [37] showed that ATF4 and other ER stress-associated molecules were markedly upregulated by TGF-β stimulation in renal tubular cells. Additionally, it was demonstrated by Verma and Datta [38] that ATF4 and other ER stress-associated molecules were induced by IL-1β via JNK in human pancreatic epithelial cells. Our results in this study are consistent with these reports that ATF4 expressions were upregulated with TGF-β or IL-1β stimulation in THP-1 macrophages, but not in HCECs. e expression of ATF4 in THP-1 macrophages was more sensitive to stimuli than in HCECs.
Here, we observed that the expression of ATF4 upon A. fumigatus infection was demonstrated to be TLR4 and LOX-1 dependent. As a toll-like receptor, TLR4 activates an intracellular signaling pathway and induces inflammatory cytokine expression as a part of innate immunity [39,40]. Yang et al. [41] demonstrated that the p-IRE1α/p-JNK/ CHOP/GRP78/ATF4 pathway that mediates ER stress was triggered by LPS-induced TLR4 activation enhancing the infiltration of monocytes and macrophages in acute renal failure. As a member of the C-type lectin super family, LOX-1 is a multiligand receptor which binds to activated platelets, apoptotic cells, C-reactive protein, and bacteria [42]. Taken together, these observations indicate that ATF4 expression may be induced by PRRs activation, including toll-like receptors and C-type lectin receptors.
In addition, we observed that ERK1/2 and JNK contributed to ATF4 expression in response to A. fumigatus infection. ERK1/2 and JNK are MAPK members which play important roles in the cellular response to proinflammatory cytokines [35,[43][44][45]. ATF4 phosphorylation was shown by Li et al. [46] to be induced by BMP-2/COX-2/PGE2 signaling pathway as part of the EP4-ERK1/2-RSK2 axis in chondrocytes. Furthermore, Lin et al. [47] demonstrated that ATF4 and other ER stress-associated molecules were abolished by the addition of a JNK inhibitor (SP600125) in protodioscin-treated cervical cancer cells. Consistent with our results, these reports from the literature indicate that ATF4 expression may be dependent on MAPKs activation.
In conclusion, our results demonstrate ATF4 was involved in the host antifungal immune response to A. fumigatus keratitis. We also demonstrated that such a response was dependent on the expression of the TLR4, LOX-1, and MAPKs pathway. ese findings were supported by a range of experimental models including A. fumigatus keratitis mice models and cell line experiments.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.