Dataset Information

Inhibition of LOX-1 alleviates the proinflammatory effects of high-mobility group box 1 in Aspergillus fumigatus keratitis.

ABSTRACT: AIM:To investigate the inflammatory amplification effect of high-mobility group box 1 (HMGB1) in Aspergillus fumigatus (A. fumigatus) keratitis and the relationship between lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) and HMGB1 in keratitis immune responses. METHODS:Phosphate buffer saline (PBS), and Boxb were injected into BALB/c mice subconjunctivally before the corneas were infected with A. fumigatus. RAW264.7 macrophages and neutrophils were pretreated with PBS and Boxb to determine the HMGB1 inflammatory amplification effects. Abdominal cavity extracted macrophages were pretreated with Boxb and Poly (I) (a LOX-1 inhibitor) before A. fumigatus hyphae stimulation to prove the the relationship between the two molecules. LOX-1, interleukin-1? (IL-1?), tumor necrosis factor-? (TNF-?), macrophage inflammatory protein-2 (MIP-2) and IL-10 were assessed by polymerase chain reaction and Western blot. RESULTS:Pretreatment with Boxb exacerbated corneal inflammation. In macrophages and neutrophils, A. fumigatus induced LOX-1, IL-1?, TNF-? and MIP-2 expression in Boxb group was higher than those in PBS group. Poly (I) treatments before infection alleviated the proinflammatory effects of Boxb in abdominal cavity extracted macrophages. Pretreatment with Boxb did not influence Dectin-1 mRNA levels in macrophages and neutrophils. CONCLUSION:In fungal keratitis, HMGB1 is a proinflammatory factor in the first line of immune response. HMGB1 mainly stimulates neutrophils and macrophages to produce inflammatory cytokines and chemokines during the immune response. LOX-1 participates in HMGB1 induced inflammatory exacerbation in A. fumigatus keratitis.

SUBMITTER: Jiang JQ

PROVIDER: S-EPMC6580210 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Inhibition of LOX-1 alleviates the proinflammatory effects of high-mobility group box 1 in <i>Aspergillus fumigatus</i> keratitis.

Jiang Jia-Qian JQ Li Cui C Cui Cong-Xian CX Ma Yu-Na YN Zhao Gui-Qiu GQ Peng Xu-Dong XD Xu Qiang Q Wang Qian Q Zhu Guo-Qiang GQ Li Chen-Yu CY

International journal of ophthalmology 20190618 6

<h4>Aim</h4>To investigate the inflammatory amplification effect of high-mobility group box 1 (HMGB1) in <i>Aspergillus fumigatus</i> (<i>A. fumigatus</i>) keratitis and the relationship between lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) and HMGB1 in keratitis immune responses.<h4>Methods</h4>Phosphate buffer saline (PBS), and Boxb were injected into BALB/c mice subconjunctivally before the corneas were infected with <i>A. fumigatus.</i> RAW264.7 macrophages and neutrophils ...[more]

PMID: 31236343

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Project description:AimTo determine whether lectin-like ox-LDL receptor (LOX-1) regulates adhesion molecules expression and neutrophil infiltration in Aspergillus fumigatus (A. fumigatus) keratitis of C57BL/6 mice.MethodsC57BL/6 mice were pretreated with a neutralizing antibody to LOX-1 (5 µg/5 µL) or control nonspecific IgG (5 µg/5 µL), LOX-1 inhibitor Poly-I (2 µg/5 µL) or PBS by subconjunctival injection. Fungal keratitis (FK) mouse models of C57BL/6 mice were established by scraping corneal central epithelium, smearing A. fumigatus on the corneal surface and covering the eye with contact lenses. The corneal response to infection was assessed via clinical score. The mRNA levels of the adhesion molecules intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin and E-selectin were tested in control and infected corneas by reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of ICAM-1 were evaluated by immunofluorescence (IF) and Western blot. Neutrophils were extracted from the abdominal cavity of C57BL/6 mice followed by pretreatment using antibody to LOX-1 (10 µg/mL) or control nonspecific IgG (10 µg/mL), the Poly-I (4 µg/mL) or PBS. The cells were then stimulated with A. fumigatus and tested mRNA and protein levels of lymphocyte function-associated antigen-1 (LFA-1) using RT-PCR and Western blot. IF and myeloperoxidase (MPO) assays were used to assess neutrophil infiltration in mice corneas.ResultsPretreatment of LOX-1 antibody or the Poly-I reduced the degree of inflammation of cornea and decreased the clinical FK score compared with pretreatment of IgG or PBS (both P<0.01). And these pretreatment also displayed an obvious decline in the mRNA levels of ICAM-1, VCAM-1, P-selectin, E-selectin and LFA-1 expression compared with control groups (all P<0.01). Furthermore, pretreated with LOX-1 antibody or Poly-I, the protein levels of ICAM-1 and LFA-1 also decreased compared with control groups (all P<0.05). Neutrophil infiltration in the cornea was significantly reduced after pretreatment of LOX-1 antibody or Poly-I compared with control groups by IF and MPO assays (both P<0.01).ConclusionInhibition of LOX-1 can decrease the expression of adhesion molecules and reduce neutrophil infiltration in A. fumigatus infected corneas of C57BL/6 mice.

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

Inhibition of LOX-1 alleviates the proinflammatory effects of high-mobility group box 1 in Aspergillus fumigatus keratitis.

Publications

Inhibition of LOX-1 alleviates the proinflammatory effects of high-mobility group box 1 in <i>Aspergillus fumigatus</i> keratitis.

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