Dataset Information

Expression and role of calcitonin gene-related peptide in mouse Aspergillus fumigatus keratitis.

ABSTRACT: AIM:To investigate the expression and role of calcitonin gene-related peptide (CGRP) in the mouse models induced by Aspergillus fumigatus (A. fumigatus). METHODS:C57BL/6 mice were randomized into a control group and A. fumigatus keratitis group. The cornea photography was assessed under the slit lamp and the clinical score was recorded after infection. Western blot, real-time polymerase chain reaction (PCR) and immunohistofluorescence analysis were applied to detect CGRP expression in cornea of both groups. In vitro, tests were conducted with C57BL/6 mice macrophages to investigate CGRP expression after interaction with A. fumigatus. Cytokines expression induced by exogenous CGRP and the antagonist CGRP8-37 in A. fumigatus-exposed macrophages was evaluated by real-time PCR and ELISA. RESULTS:The cornea expression of CGRP was significantly elevated in C57BL/6 mice corneas and macrophages after A. fumigatus infection. After treatment with exogenous CGRP, the levels of interleukin-1? (IL-1?), tumor necrosis factor-? (TNF-?) and IL-6 were reduced, and IL-10 level was increased in the A. fumigatus stimulated-macrophages. However, IL-1?, TNF-? and IL-6 levels were upregulated after pretreatment of CGRP8-37. But the mRNA levels of MIP-2, TGF-? and IL-10 were not changed. CONCLUSION:This study provides evidence that A. fumigatus increased CGRP expression. CGRP may play a protective role against inflammation in A. fumigatus keratitis.

SUBMITTER: Yin M

PROVIDER: S-EPMC6520267 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Expression and role of calcitonin gene-related peptide in mouse <i>Aspergillus fumigatus</i> keratitis.

Yin Min M Li Cui C Peng Xu-Dong XD Zhao Gui-Qiu GQ Wu Yuan Y Zheng Heng-Rui HR Wang Qian Q Xu Qiang Q Jiang Nan N

International journal of ophthalmology 20190518 5

<h4>Aim</h4>To investigate the expression and role of calcitonin gene-related peptide (CGRP) in the mouse models induced by <i>Aspergillus fumigatus</i> (<i>A. fumigatus</i>).<h4>Methods</h4>C57BL/6 mice were randomized into a control group and <i>A. fumigatus</i> keratitis group. The cornea photography was assessed under the slit lamp and the clinical score was recorded after infection. Western blot, real-time polymerase chain reaction (PCR) and immunohistofluorescence analysis were applied to ...[more]

PMID: 31131225

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Project description:AIM:To observe the expression and role of aryl hydrocarbon receptor (AhR) in the immune response of mouse cornea infected with Aspergillus fumigatus (A. fumigatus). METHODS:Murine models of A. fumigatus keratitis were established by scraping the central epithelium of mouse cornea, daubing A. fumigatus on the cornea and covering with a contact lens. The mice were randomly divided into the control group and the A. fumigatus-infected (A.F.) group for 1, 3 and 5d respectively, which corneas were daily monitored by a slit lamp microscope and the clinical scores were also recorded timely after infection. In this study, immunofluorescence staining was used to detect the expression and localization of AhR in mouse corneas, and the mRNA and protein of AhR were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. In addition, mouse peritoneal macrophages were stimulated by A. fumigatus with or without the pretreatment of AhR antagonist CH223191 and AhR agonist FICZ, and the tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10) and Arg-1 mRNA were detected by RT-PCR. RESULTS:According to the results of the slit light photography, it was clearly indicated that the corneal inflammation were the most severe and the clinical score became the highest as well on the 3rd day after the infection of A. fumigatus. Contrasted with the control group, the expression of AhR in the corneal epithelial cells infected with A. fumigatus was significantly increased detected by immunofluorescence staining. AhR mainly expressed in the nucleus and cytoplasm of corneal epithelial cells. Consistent with the transcriptional level of AhR mRNA, the expression level of AhR protein reached the peak on the 3rd day after infection which was detected by Western blot. Furthermore, RT-PCR showed that CH223191 up-regulated the expression of TNF-α and iNOS and down-regulated the expression of IL-10 and Arg-1 in peritoneal macrophages; inversely, FICZ reduced the expression of TNF-α and iNOS while elevated the expression of IL-10 and Arg-1. CONCLUSION:AhR is involved in the pathogenesis of A. fumigatus keratitis and induced immune protection in anti-A. fumigatus immune response by inhibiting M1 and increasing M2 phenotype macrophage-related inflammatory factors.

Project description:AimTo investigate the expression of macrophage migration inhibitory factor (MIF) and detect its role in the innate immune response of fungal keratitis (FK).MethodsWe collected the paraffin-embedded cornea tissues from 10 FK and 6 ocular trauma patients to explore the MIF expression by immunohistochemistry. Then we cultured telomease-immortalized human corneal epithelial cells (THCEs), stimulated by the hyphae suspension of Aspergillus fumigatus (A. fumigatus) to detect the change of MIF with or without the pretreatment of MIF inhibitor [4-Iodo-6-phenylpyrimidine (4-IPP)] by real-time polymerase chain reaction (PCR). The protein level of MIF was also tested by immunohistochemistry, and the level of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA were compared between normal, hyphae stimulated and 4-IPP pretreated groups by real-time PCR to study the influence of MIF on the expression of TNF-α and IL-6. Corneal severity of rats' FK models was documented by clinical scores, and real-time PCR. Western blot and immunohistochemistry were used to test the expression of MIF, TNF-α and IL-6 in rats' corneas.ResultsIn the corneas of FK patients, there was much stronger expression of MIF than that in the normal group showed by immunohistochemistry. In cultured THCEs stimulated by A. fumigatus, the expression of MIF became stronger in both immunohistochemistry and PCR at 16, 24, 32 and 48h post infection (p.i.; P<0.01, P<0.01, P<0.01, P<0.05). After pretreated with 4-IPP, the expression of MIF reduced at 4, 8, 16h p.i. (P<0.05, P<0.05, P<0.05) and the downstream TNF-α and IL-6 decreased obviously (P<0.05, P<0.01). In rats with A. fumigatus keratitis, the relative mRNA and protein level of MIF increased than those in the normal group by PCR (at 1d: P<0.01, 3d: P<0.01, 5d: P<0.01), Western blot and immunohistochemistry. After blocked MIF with 4-IPP, the clinical outcomes of rat keratitis showed markedly reduced inflammatory response (P<0.01), with TNF-α and IL-6 decreased in accordance with those in THCEs by PCR (P<0.05, P<0.01).ConclusionThe expression of MIF increased significantly in FK patients, THCEs and rats stimulated by A. fumigatus. After blocked with 4-IPP, the expression of MIF reduced, and so did its downstream cytokines: TNF-α and IL-6. The inflammation reaction of the rats' corneas lightened after pretreated with 4-IPP. MIF may play a role in the innate immune response of the corneal resistance against A. fumigatus.

Dataset Information

Expression and role of calcitonin gene-related peptide in mouse Aspergillus fumigatus keratitis.

Publications

Expression and role of calcitonin gene-related peptide in mouse <i>Aspergillus fumigatus</i> keratitis.

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