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

Neutrophil Caspase-11 Is Required for Cleavage of Caspase-1 and Secretion of IL-1β in Aspergillus fumigatus Infection.

Neutrophils are an important source of IL-1β secretion in bacterial infections, where they infiltrate affected tissues in log-fold higher numbers than macrophages. Neutrophils also have functional NLRP3 and NLRC4 inflammasomes that can process pro-IL-1β to the bioactive 17-kDa form. In the current study, we examined the role of IL-1β in response to corneal infection with the filamentous fungus Aspergillus fumigatus and found that neutrophils were the predominant source of bioactive IL-1β in the cornea. We also observed that caspase-11-/- mice exhibit the same susceptibility phenotype as IL-1β-/-, ASC-/-, NLRP3-/-, and caspase-1-/- mice, with impaired neutrophil recruitment to infected corneas and increased hyphal growth. We further demonstrate that caspase-11 is required for caspase-1 activation and IL-1β processing during infection. In vitro, we show that caspase-11 is regulated by the common type I IFN receptor (IFNAR) through JAK-STAT signaling and that caspase-11 is required for speck formation and caspase-1 activity. Aspergillus spores (conidia) stimulate IL-1β processing and secretion in neutrophils activation of Dectin-1 and signaling through the Raf1 kinase/MEKK rather than the spleen tyrosine kinase pathway. Collectively, these findings reveal unexpected regulation of IL-1β production by neutrophils in response to pathogenic fungi

1-MT treatment enhanced inflammatory cytokine production in corneas of mice infected with <i>A</i>. <i>fumigatus</i>.

<p>C57BL/6 mice were inoculated with 2 μl of 1 × 10⁵/μl spores. (A) The corneas were monitored and taken photograps under a slit lamp, and (B) evaluated with the scoring system. Disease score is shown as mean ± standard deviation. After 1-MT treatment, the IL-1β mRNA and protein levels were significantly increased (C, D) at 3 days after infection in 1-MT treated mice compared with PBS-treated mice. In addition, both IL-6 mRNA (E) and IL-6 protein (F) were significantly increased at 3 days after infection. In addition, the IL-1β proteins were increased at 5 days after infection in 1-MT treated mice. (*<i>p</i> < 0.05, **<i>p</i> < 0.001 compared with PBS treated mice).</p

IDO expression in cultured telomerase-immortalized HCECs infected with <i>A</i>. <i>fumigatus</i>.

<p>Relative expression of IDO mRNA was detected by qRT-PCR 4, 10, 16 and 24 h after incubation with <i>A</i>. <i>fumigatus</i> spores at three different concentrations (5 × 10⁶ /ml, 5 × 10⁷ /ml and 5 × 10⁸ /ml). IDO expression increased by 4- to 5-fold with a peak level at 10 h after exposure to inactivated <i>A</i>. <i>fumigatus</i> spores (5 × 10⁷ /ml).</p

Indoleamine 2,3-Dioxygenase Is Involved in the Inflammation Response of Corneal Epithelial Cells to <i>Aspergillus fumigatus</i> Infections

<div><p>Indoleamine 2,3-dioxygenase (IDO), which is mainly expressed in activated dendritic cells, is known as a regulator of immune responses. However, the role of IDO in immune responses against fungal corneal infection has not been investigated. To evaluate the regulatory mechanisms of IDO in fungal inflammation, we resorted to human corneal epithelial cells (HCECs), known as the first barrier of cornea against pathogenic microorganisms. We found that IDO was significantly up-regulated in corneal epithelium infected with <i>Aspergillus fumigatus</i> (<i>A</i>. <i>fumigatus</i>) and HCECs incubated with spores of <i>A</i>. <i>fumigatus</i>. Furthermore, IDO inhibitor (1-methyltryptophan, 1-MT) enhanced inflammatory cytokines IL-1β and IL-6 expression which were up-regulated by <i>A</i>. <i>fumigatus</i> spores infection. Dectin-1, as one of the important C-type lectin receptors, can identify β-glucan, and mediate fungal innate immune responses. In the present study, pre-treatment with curdlan, a Dectin-1 agonist, further enhanced IDO expression compared with <i>A</i>. <i>fumigatus</i> stimulation. While laminarin, the Dectin-1 specific inhibitor, partially inhibited IDO expression stimulated by <i>A</i>. <i>fumigatus</i>. Further studies demonstrated inhibition of IDO activity amplified the expressions of inflammatory cytokines IL-1β and IL-6 induced by activation of Dectin-1. These results suggested that IDO was involved in the immune responses of fungal keratitis. The activation of Dectin-1 may contribute to <i>A</i>. <i>fumigatus</i> spores-induced up-regulation of IDO.</p></div

Expression of IDO mRNA in human corneal epithelium with fungal infection.

<p>qRT-PCR was performed to identify relative expression of IDO mRNA in corneal epithelium. Normal corneal tissue remainings after corneal transplantation were considered controls. Corneas with fungal infections were classified into three grades (mild, moderate and severe) according to keratomycosis severity. Fungal infection increased IDO mRNA expression in the human corneal epithelium and the expression correlated with keratomycosis severity. The data were represented as the mean ± standard deviation of four independent experiments. (**<i>p</i> < 0.01, ***<i>p</i> < 0.001 compared with control; ^##<i>p</i> < 0.01, compared with mild grade group; ^$$ $<i>p</i> < 0.001 compared with moderate grade group).</p

The effect of Dectin-1 on IDO expressions in cultured HCECs infected with <i>A</i>. <i>fumigatus</i>.

<p>The HCECs were pre-treated with curdlan or laminarin, and were exposed to inactivated <i>A</i>. <i>fumigatus</i> spores for 10 h. The cultures were subjected to qRT-PCR to measure IDO mRNA expressions (A). The cultures treated for 24 h were used to evaluate IDO protein expressions in HCECs by western blot (B). The data are represented as mean ± standard deviation of four independent experiments. (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p