Fumigatus Keratitis Research Articles

Efficacy of adjuvant intrastromal and combination of intrastromal and intracameral voriconazole in <em>Aspergillus fumigatus</em>-induced moderate fungal keratitis in rabbits

BACKGROUND There is no in vivo evidence for the effectiveness of adjuvant intrastromal and combination of intrastromal and intracameral voriconazole (VCZ) for treating Aspergillus fumigatus keratitis. This study aimed to compare the efficacy of both agents against it. 
 METHODS A randomized, masked, controlled experimental study was conducted on 11 albino New Zealand white rabbits in which moderate fungal keratitis was induced by inoculating spores of A. fumigatus to the cornea. The rabbits were allocated into 3 groups: 50 μg/0.1 ml intrastromal VCZ injection, 50 μg/0.1 ml intrastromal VCZ and intracameral VCZ injections, and topical VCZ (control). The treatment was given 5 days after inoculation. Epithelial defect, infiltrate size, corneal ulcer depth, and hypopyon were evaluated clinically. Histopathological and mycological examinations were also done 14 days after treatment. 
 RESULTS All rabbits in the adjuvant treatment groups demonstrated a tendency of a better clinical response with decreasing size of epithelial defect (p = 0.679) and infiltrate (p = 0.755) than in the control group. Direct microscopy, corneal culture, and chop corneal tissue culture were still positive in most of the rabbits from all groups. Histopathological examination showed an increase of inflammatory cells after treatment in all groups, especially in rabbits which were inoculated with A. fumigatus spores in both eyes. 
 CONCLUSIONS An adjuvant combination of intrastromal and intracameral VCZ showed a tendency of better clinical response for A. fumigatus-induced moderate fungal keratitis in rabbits.

Quercetin ameliorates Aspergillus fumigatus keratitis by inhibiting fungal growth, toll-like receptors and inflammatory cytokines

PurposeTo investigate the antifungal and anti-inflammatory effects of quercetin on Aspergillus fumigatus (A. fumigatus) keratitis. MethodsHuman corneal epithelial cells (HCECs) and C57BL/6 mice were stimulated by A. fumigatus and treated with quercetin or dimethyl sulfoxide (DMSO) after infection. In HCECs, minimum inhibitory concentration (MIC) and cytotoxicity tests (CCK-8) were used to detect the antifungal effect and cytotoxicity of quercetin. In mice with A. fumigatuskeratitis, clinical score, plate counting and hematoxylin-eosin (HE) staining were performed to evaluate the effects of quercetin in vivo. Myeloperoxidase (MPO) assay and immunofluorescence staining were applied to assess neutrophil recruitment and infiltration. Real time PCR (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and western blot were used to detect the mRNA and protein expressions of inflammatory mediators. ResultsCompared with DMSO control, quercetin (16–64 μM) significantly inhibited the growth of A. fumigatus in a concentration-dependent manner without affecting cell viability in HCECs. In corneas of mice with A. fumigatuskeratitis, quercetin decreased clinical score and fungal load, and reduced neutrophil recruitment and infiltration to the corneal stroma. Moreover, quercetin attenuated the expression of inflammatory mediators including toll-like receptor-4 (TLR-4), TLR-2, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and high mobility group box 1 (HMGB1) in vitro and in vivo. ConclusionsOur study demonstrated that quercetin treatment can ameliorate A. fumigatus keratitis by inhibiting the growth of A. fumigatus, decreasing neutrophil recruitment and infiltration, and downregulating the productions of TLR-4, TLR-2, TNF-α, IL-1β and HMGB1, indicating quercetin is likely to become a potential therapeutic agent in FK treatment.

CD23 mediated the induction of pro-inflammatory cytokines Interleukin-1 beta and tumor necrosis factors-alpha in Aspergillus fumigatus keratitis.

CD23 mediated the induction of pro-inflammatory cytokines Interleukin-1 beta and tumor necrosis factors-alpha in Aspergillus fumigatus keratitis.

TSLP Produced by Aspergillus fumigatus-Stimulated DCs Promotes a Th17 Response Through the JAK/STAT Signaling Pathway in Fungal Keratitis.

PurposeThe purpose of this study was to explore the role of thymic stromal lymphopoietin (TSLP) secreted by Aspergillus fumigatus–stimulated dendritic cells (DCs) during the T helper 17 (Th17) immune response, and further clarify the mechanisms contributing to the Th17 immune response of fungal keratitis (FK).MethodsA carboxyfluorescein diacetate succinimidyl ester assay, PCR, and flow cytometry were performed to detect Th17 differentiation of CD4+ T cells; PCR, ELISA, and Western blot were used to detect the expression of TSLP and JAK/STAT–related proteins; Signaling pathways involved in Th17 response was evaluated using RNA sequence; C57BL/6 mice were infected with A. fumigatus and treated with ruxolitinib or BBI608. Slit-lamp examination, fluorescein staining, and clinical scores were used to assess the clinical manifestation.ResultsA. fumigatus–infected DCs could drive naïve CD4+ T-cell proliferation and promote the production of Th17 cytokines IL-17A, IL-17F, and IL-22. A. fumigatus stimulation increased the expression of TSLP in DCs. DC-derived TSLP contributed to a Th17-type inflammatory response via the JAK/STAT signaling pathway. TSLP small interfering RNA, TSLPR small interfering RNA, or JAK/STAT inhibitors inhibited the Th17 immune response induced by A. fumigatus–infected DCs. Moreover, TSLP promoted A. fumigatus keratitis disease progression in a mouse model. However, inhibition of the JAK/STAT signaling pathway using a specific inhibitor reversed the development of FK by A. fumigatus infection.ConclusionsTSLP secreted by A. fumigatus–stimulated DCs played a significant role in the Th17-dominant immune response of FK through its JAK/STAT activation. Our findings may contribute to the elucidation of the molecular mechanisms of FK and to the development of novel therapeutic approaches.

GSDMD, an executor of pyroptosis, is involved in IL-1β secretion in Aspergillus fumigatus keratitis

The protein GSDMD is an important performer of pyroptosis and a universal substrate for the inflammatory caspase. However, the role and regulatory mechanism of GSDMD in Aspergillus fumigatus keratitis is remains unknown. Here we detected GSDMD protein in the cornea of normal and fungal-infected C57BL/6 mice. Human corneal epithelial cell (HCECs) were preincubated with a hydrochloride solution (IFNR inhibitor), ruxolitinib (JAK/STAT inhibitor), belnacasan (caspase-1 inhibitor) before infection with A. fumigatus conidia. Mice corneas were infected with Aspergillus fumigatus after pretreatment of GSDMD siRNA via subconjunctival injection. After, samples were harvested at specific time points and the expression of GSDMD and IL-1β was assessed by PCR, Western blot and immunofluorescence staining. Compared with the control group, we observed that the expression of GSDMD in fungal-infected mice cornea was significantly increased. After pretreatment with IFNR, JAK/STAT and caspase-1 inhibitors before fungal infection, the expression of GSDMD was significantly inhibited compared to the DMSO control in HCECs. Moreover, the GSDMD siRNA treatment have significantly weaken corneal inflammatory response, decreasing the proinflammatory factor IL-1β secretion and reducing neutrophils and macrophages recruitment in mice infected corneas. In summary, the data here provided evidences that GSDMD, an executor of pyroptosis, is involved in the early immune response of A. fumigatus keratitis. Additionally, the inhibition of GSDMD expression can affect the secretion of IL-1β and the recruitment of neutrophil and macrophages by blocking IFNR, JAK/STAT and caspase-1 signaling pathway. The protein GSDMD may emerge as a potential therapeutic target for A. fumigatus keratitis.

The cGAS-STING signaling pathway contributes to the inflammatory response and autophagy in Aspergillus fumigatus keratitis

Fungal keratitis is a serious corneal infection, which can lead to significant visual impairment and blindness. The cGAS-STING signaling pathway has emerged as a key player in innate immunity by sensing of invading pathogens. However, the role of the cGAS-STING pathway in Aspergillus fumigatus (A. fumigatus) keratitis is still unknown. In this study, we showed that the cGAS-STING signaling pathway was activated in human corneal epithelial cells (HCECs) and in mouse corneas infected with A. fumigatus. Knockdown of cGAS reduced A. fumigatus-induced production of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IFN-β. However, reconstruction of cGAS activity restored the inflammatory response in HCECs infected with A. fumigatus. A specific cGAS inhibitor, RU.521, could also significantly inhibit A. fumigatus-induced inflammatory cytokine expression. In addition, we found that cGAS was indispensable for the autophagy flux evoked by A. fumigatus infection. Moreover, inhibition of cGAS using siRNA or RU.521 alleviated the severity of A. fumigatus keratitis in the mouse cornea. Therefore, the cGAS-STING signaling pathway contributes to the progression of A. fumigatus keratitis and targeting this pathway may provide therapeutic potential.

Anti-inflammatory effects of astaxanthin against fungal keratitis.

To characterize effect of astaxanthin (ASX) in Aspergillus fumigatus (A. fumigatus) induced keratitis in mouse model. In vivo, fungal keratitis mouse model was established in C57BL/6 mice using A. fumigatus, followed by ASX or dimethyl sulfoxide (DMSO) treatment. Clinical responses were evaluated by clinical score and myeloperoxidase (MPO) assay. Inflammatory cytokines were assessed by reverse-transcription polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, and enzyme-linked immuno sorbent assay (ELISA). In animal model, ASX improved corneal transparency and clinical response, suppressed the expression of inflammatory cytokine like IL-1β, TNF-α, and HMGB-1. Neutrophil levels have been shown to decrease in ASX-treated cornea by immunofluorescence and MPO. TLR2 and TLR4 levels were lower in ASX-treated group than DMSO-treated. ASX can suppress inflammatory response and reduce inflammatory cytokine production in mice model with A. fumigatus keratitis.

Mammalian Ste20-like kinase 4 inhibits the inflammatory response in Aspergillus fumigatus keratitis

Mammalian Ste20-like kinase 4 (MST4), a new member of the germinal-center kinase STE20 family, was recently demonstrated to be a negative regulator of inflammation. However, whether MST4 participates in the inflammatory response to fungal infection remains unknown. Our study investigated the role and molecular mechanisms of MST4 in mice cornea and corneal epithelial cells exposed to Aspergillus fumigatus (A. fumigatus). Protein level of MST4 was detected in mice corneas and human corneal epithelial cells (HCECs) by Western blot analysis. The MST4 protein level was significantly elevated in mice corneas infected with A. fumigatus and HCECs exposed to A. fumigatus. MST4 expression was also detected in mice corneas by immunofluorescence staining. Furthermore, we found recombinant MST4 inhibited proinflammatory cytokines expressions induced by A. fumigatus at both the mRNA and protein levels in mice corneas and HCECs. To further investigate the mechanism of MST4′s anti-inflammatory effect in A. fumigatus keratitis, we verified recombinant MST4 can inhibit curdlan-mediated proinflammatory cytokines production in HCECs. Surprisingly, recombinant MST4 protein downregulated A. fumigatus-induced Dectin-1 expression in both mRNA and protein levels in mice corneas. Recombinant MST4 can inhibit the mRNA expression level of Dectin-1 which was induced by curdlan in HCECs. MST4 can also inhibit the expression of Dectin-1 in mRNA levels increased by Dectin-1 overexpression plasmid in HCECs. Moreover, A. fumigatus or curdlan significantly induced the phosphorylation of Syk, which was consequently suppressed by recombinant MST4. Finally, recombinant MST4 promotes HCECs proliferation, which contribute to cornea wound healing. Taken together, our results provide evidences that MST4 inhibits inflammatory signaling response in A. fumigatus keratitis by downregulating Dectin-1/p-Syk pathway and simultaneously promotes HCECs proliferation.

Lacrimal androgen-binding proteins protect against Aspergillus fumigatus keratitis in mice

AimTo clarify the regulatory mechanisms of lacrimal androgen-binding proteins (ABPs) in mice with keratitis caused by Aspergillus fumigatus (A. fumigatus). MethodsMouse models of A. fumigatus keratitis were established. Lacrimal glands were removed after 24 h for general and histological comparison. Lacrimal ABPs were detected by qRT-PCR and quantitative proteomic analysis, or were detected by qRT-PCR after subconjunctival or lacrimal gland injection with dexamethasone. Unique inflammatory factors were detected by qRT-PCR, Western blot and/or immunofluorescence. Interleukin-1β (IL-1β) was injected into the lacrimal gland to explore the relationship between IL-1β and lacrimal ABPs. ResultsThe lacrimal glands of mice with fungal keratitis were larger than normal mice and these structures became disorganized. Moreover, the expression of ABP ε and ABP δ were increased. Subconjunctival injection with dexamethasone could reduce the size of the lacrimal gland and increase the expression of ABP ε and ABP δ, while lacrimal gland injection with dexamethasone had no obvious effects. The expression of IL-1β in the lacrimal gland of mice with A. fumigatus keratitis was increased. When IL-1β was injected into the lacrimal gland, the lacrimal gland enlarged and the expression of ABP ε and ABP δ decreased. ConclusionLacrimal glands contributed to protection in fungal keratitis, which was not due to the involvement of inflammatory cells in mice. ABP δ and ABP ε of mice were involved in reducing the severity of corneal damage in mice with A. fumigatus keratitis. Moreover, the expression of IL-1β and ABP δ and ABP ε were intrinsically linked.

Indoleamine 2,3-Dioxygenase Regulates Macrophage Recruitment, Polarization and Phagocytosis in Aspergillus Fumigatus Keratitis.

PurposeTo explore the influence of indoleamine 2,3-dioxygenase (IDO) on macrophage recruitment, polarization and phagocytosis in Aspergillus fumigatus keratitis.MethodsA murine model of A. fumigatus keratitis and peritoneal macrophages incubated with the hyphae of A. fumigatus were used. Macrophage recruitment in corneas was evaluated using immunofluorescence staining. The polarization of macrophages, which was stimulated by A. fumigatus and pretreatment with or without 1-methyltryptophan (1-MT), interferon gamma (IFNG), extracellular regulated protein kinases (ERK) antagonist, and p38 antagonist, was determined using reverse-transcription polymerase chain reaction and flow cytometry. P38 and ERK levels were determined using Western blotting. Macrophage phagocytosis was examined using colony-forming units.ResultsCompared with the A.F. group, recruitment of macrophages increased, tumor necrosis factor–α (TNF-α) and inducible nitric oxide synthase (iNOS) expression decreased, whereas arginase-1 (Arg-1) and interleukin-10 (IL-10) expression increased in the mouse corneas of the 1-MT+A.F. group. The ratio of CD206+/CD86+ macrophages in the corneas and spleens of 1-MT+A.F. group increased. Furthermore, in peritoneal macrophages stimulated by A. fumigatus, 1-MT promoted Arg-1 and IL-10 expression while upregulating the ratio of CD206+/CD86+ macrophages. Conversely, IDO agonist IFNG promoted TNF-α and iNOS expression, inhibited Arg-1 and IL-10 expression and downregulated the ratio of CD206+/CD86+ macrophages. The role of IFNG was reversed by the antagonist of P38 or ERK. P38 and ERK levels were downregulated in corneas of 1-MT+A.F. group. Besides, IFNG inhibited macrophage phagocytosis.ConclusionsIDO inhibited macrophage recruitment and phagocytosis in A. fumigatus keratitis. Mechanistically, IDO is involved in M1 macrophage polarization in A. fumigatus keratitis through a MAPK/ERK-dependent pathway.

Perillaldehyde Ameliorates Aspergillus fumigatus Keratitis by Activating the Nrf2/HO-1 Signaling Pathway and Inhibiting Dectin-1-Mediated Inflammation.

PurposeThe purpose of this study was to investigate the therapeutic effect of perillaldehyde (PAE) on Aspergillus fumigatus (A. fumigatus) keratitis.MethodsHuman corneal epithelial cells (HCECs) were pretreated with PAE and stimulated with A. fumigatus mycelium. C57BL/6 mice were infected with A. fumigatus and treated with or without PAE 1 day after infection. Clinical scores, PCR, ELISA, and Western blotting were used to detect the expression of pro-inflammatory mediators, dendritic cell-associated c-type lectin-1 (Dectin-1), nuclear factor (erythroid-derived 2) like 2 (Nrf2), and heme oxygenase (HO-1). Nrf2 expression in HCECs pretreated with PAE was observed by immunofluorescence. NIMP-R14 protein expression and localization in mouse corneas were observed by immunofluorescence staining after treatment with PAE. Corneal colony counting, time-kill tests, and mycelial transformation inhibition tests were used to evaluate the antifungal effect of PAE.ResultsC57BL/6 mice treated with PAE at 1 day after infection had a lower clinical score and decreased IL-1β, TNF-α, IL-6, Dectin-1, and MPO levels. PAE treatment significantly reduced neutrophil recruitments to the corneal stroma. Compared with the DMSO-treated group, PAE treatment significantly decreased mRNA and protein levels of pro-inflammatory cytokines and Dectin-1 in HCECs. PAE pretreatment before A. fumigatus stimulation obviously elevated the mRNA and protein levels of components of the Nrf2/HO-1 axis. HCECs pretreated with PAE before infection showed a weakened ability to inhibit inflammation in the presence of brusatol (BT; an Nrf2 inhibitor) or ZnPP (an HO-1 inhibitor). PAE treatment significantly reduced the fungal load of C57BL/6 mouse corneas and inhibited fungal growth in vitro.ConclusionsThese data proved that PAE may ameliorate A. fumigatus keratitis by activating the Nrf2/HO-1 signaling pathway and inhibiting the Dectin-1 mediated inflammatory response and neutrophil recruitment. Furthermore, PAE exerts direct fungicidal activity on A. fumigatus.

Regulation of LOX-1 on adhesion molecules and neutrophil infiltration in mouse Aspergillus fumigatus keratitis.

To 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. C57BL/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. Pretreatment 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). Inhibition of LOX-1 can decrease the expression of adhesion molecules and reduce neutrophil infiltration in A. fumigatus infected corneas of C57BL/6 mice.

The anti-inflammatory regulation of calcitonin gene-related peptide in mouse Aspergillus fumigatus keratitis.

To analyze the impact of calcitonin gene-related peptide (CGRP) in mouse keratitis after Aspergillus fumigatus (A. fumigatus) infection. C57BL/6 mice were treated subconjunctivally with different concentrations of exogenous CGRP, and BALB/c mice were treated with CGRP8-37 (a CGRP antagonist) before corneas were infected with A. fumigatus. The cornea was assessed under the slit-lamp and the clinical score was recorded. The mRNA levels of IL-1β, TNF-α, IL-6, and MIP-2 were detected by quantitative real-time polymerase chain reaction (PCR), while the protein level of IL-1β was determined by Western blotting. In vitro, RAW264.7 cells were used to investigate NLRP3 and IL-1β expression induced by A. fumigatus after the pretreatment of exogenous CGRP or CGRP8-37. Cytokines expression in RAW264.7 cells was evaluated by real-time PCR and Western blotting. Using exogenous CGRP resulted in down-regulated synthesis of IL-1β and MIP-2 stimulated by A. fumigatus in C57BL/6 mice keratitis, and the synthesis of IL-1β, MIP-2 and IL-6 was up-regulated in BALB/c mice corneas after the pretreatment with CGRP8-37. Pretreatment with exogenous CGRP and CGRP8-37 did not influence TNF-α mRNA levels either in BALB/c or C57BL/6 mice keratitis. The levels of NLRP3 and IL-1β were both reduced in A. fumigatus stimulated-macrophages after treatment with exogenous CGRP. And CGRP8-37 pretreatment would increase NLRP3 and IL-1β levels. CGRP may alleviate the inflammatory reaction in mice keratitis after infection with A. fumigatus. The anti-inflammatory effect may be related to the inhibition of NLRP3 expression by CGRP.

High-mobility group box1 as an amplifier of immune response and target for treatment in Aspergillus fumigatus keratitis.

To determine the roles of high-mobility group box1 (HMGB1) in pro-inflammation, host immune response and its potential target for treatment in Aspergillus fumigatus (A.fumigatus) keratitis. Expression of HMGB1 was tested in C57BL/6 normal and infected corneas. Dual immunostaining tested co-expression of HMGB1 with TLR4 or LOX-1. C57BL/6 mice were pretreated with Box A or PBS and then infected. Clinical scores, polymerase chain reaction, ELISA, and MPO assay were used to assess the disease response. Flow cytometry were used to test the effect of Box A on reactive oxygen species (ROS) expression after A.fumigatus stimulation in polymorphonuclear neutrophilic leukocytes (PMN). C57BL/6 peritoneal macrophages were pretreated with Box B before A.fumigatus stimulation, and MIP-2, IL-1β, TNF-α, HMGB1 and LOX-1 were measured. Macrophages were pretreated with Box B or Box B combined with Poly(I) (an inhibitor of LOX-1) before stimulating with A.fumigatus, and MIP-2, IL-1β, TNF-α, LOX-1, p38-MAPK, p-p38-MAPK were measured. HMGB1 levels were elevated in C57BL/6 mice after infection. HMGB1 co-expressed with TLR4, and LOX-1 in infiltrated cells. Box A vs PBS treated C57BL/6 mice had lower clinical scores and down-regulated corneal HMGB1, MIP-2, IL-1β expression and neutrophil influx. Box B treatment amplified expression of MIP-2, IL-1β, TNF-α, HMGB1 and LOX-1 that induced by A.fumigatus in macrophage. Compared to the treatment of Box B only, the protein expression of IL-1β, TNF-α showed inhibition of Box B combined with Poly(I), which also reduced the A.fumigatus-evoked protein level of LOX-1 and phosphorylation level of p38-MAPK. The production of A.fumigatus-stimulated ROS was significantly declined after Box A pretreatment in PMN. Blocking HMGB1 reduces the disease response in C57BL/6 mice. HMGB1 can amplify the host immune response through p38-MAPK, and is a target for treatment of A.fumigatus keratitis.

FCN-A mediates the inflammatory response and the macrophage polarization in Aspergillus fumigatus keratitis of mice by activating the MAPK signaling pathway

Fungal keratitis (FK) is a severe corneal disease that may cause vision loss. Previous studies indicate that the innate immune response produces the most effective anti-Aspergillus immune resistance. Ficolin-A (FCN-A), a soluble pattern-recognition receptor (PRR) family plays an important role in the innate immunity. In this study, we aimed to study the role of FCN-A in the A. fumigatus infected cornea. Here for the first time, we reported that the expression of FCN-A increases after A. fumigatus infection in the cornea of mice. Then, our results showed that the down-regulation of FCN-A reduced the inflammatory response of the cornea infected mice and decreased the expression of the TNF-a, p-p38, p-JNK. We also found that FCN-A can affect the recruitment of macrophages in the cornea of mice with A. fumigatus keratitis. In the mouse model of A. fumigatus keratitis and the A. fumigatus stimulation of RAW 264.7 cells, knocking down of FCN-A expression promoted the macrophage polarization toward M2. Furthermore, we observed that both the p38 and JNK inhibitors pretreatment decreased the proportion of M1/M2 in RAW 264.7 cells. Taken together, our data provide evidence that FCN-A participated in the inflammatory response of A. fumigatus keratitis in mice. Moreover, FCN-A mediates the inflammatory response and the polarization of the macrophages by activating the MAPK signaling pathway in A. fumigatus keratitis.

The Role of Autophagy in the Innate Immune Response to Fungal Keratitis Caused by Aspergillus fumigatus Infection.

PurposeTo determine the role of autophagy in the innate immune response to fungal keratitis (FK) caused by Aspergillus fumigatus infection.MethodsCorneal samples obtained from patients and mice with FK were visualized via transmission electron microscopy (TEM). Autophagy-related proteins LC3B-II, Beclin-1, LAMP-1, and p62 in A. fumigatus-infected corneas of C57BL/6 mice were tested by Western blot. After treatment with autophagy inhibitors 3-methyladenine (3-MA), chloroquine (CQ), or inducer rapamycin, autophagy-related proteins were detected by Western blot. Corneas were photographed with slit lamp microscopy and pathological changes were observed by hematoxylin and eosin staining. Polymorphonuclear neutrophilic leukocytes (PMNs) were assessed by immunofluorescent staining and observed under TEM. The levels of CXCL-1, IL-1β, HMGB1, IL-18, TNF-α, and IL-10 were tested by reverse transcription polymerase chain reaction and Western blot. The quantification of fungal loads was detected and photographed.ResultsThe accumulation of autophagosomes in corneas of patients and mice with FK was observed with TEM. The expression of LC3B-II, Beclin-1, and LAMP-1 was elevated in corneas after fungal infection, whereas p62 was reduced. Treatment with 3-MA or CQ upregulated clinical scores, pathological changes, and the expression of CXCL-1, IL-1β, HMGB1, IL-18, and TNF-α except IL-10. The morphology of PMNs was changed and PMN recruitment was increased in mice corneas treated with 3-MA or CQ, whereas rapamycin reduced the inflammatory response to keratitis. These results were statistically significant.Conclusions A. fumigatus infection increases the expression of autophagy in corneas. Autophagy plays an anti-inflammatory role in the innate immune response to A. fumigatus keratitis.

Expression and role of aryl hydrocarbon receptor in Aspergillus fumigatus keratitis.

To observe the expression and role of aryl hydrocarbon receptor (AhR) in the immune response of mouse cornea infected with Aspergillus fumigatus (A. fumigatus). 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. 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. 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.

Nerolidol inhibits the LOX-1 / IL-1β signaling to protect against the Aspergillus fumigatus keratitis inflammation damage to the cornea

PurposeNerolidol, a naturally occurring sesquiterpene has both anti-microbial and anti-inflammatory properties. The current study aims to investigate the antifungal and the anti-inflammatory effects of nerolidol against mouse Aspergillus fumigatus (A. fumigatus) keratitis. MethodsThe minimum inhibitory concentration (MIC) and cytotoxicity tests were used to study the antifungal ability. For in vivo and in vitro studies, the mouse corneas and the human corneal epithelial cells (HCECs) infected with A. fumigatus spores were intervented with nerolidol or phosphate buffer saline (PBS). Thereafter, the effect of the nerolidol on the response against inflammation was analyzed using the following parameters: recruitment of the neutrophils or macrophages and the expression of the lectin-type oxidized low density lipoprotein receptor-1 (LOX-1) and interleukin 1β (IL-1β). Techniques used were the slit lamp, immunofluorescence, myeloperoxidase (MPO) detection, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. ResultsNerolidol directly inhibits the growth of A. fumigatus. The administration of nerolidol reduced the severity of fungal keratitis with infiltration of fewer inflammatory cells and reduced levels of the LOX-1, as well the anti-inflammatory cytokines such as IL-1β were reduced compared with the PBS group. Additionally, in vitro studies showed that treatment with nerolidol inhibited the production of the LOX-1 / IL-1β levels in A. fumigatus stimulated HCECs. ConclusionNerolidol attenuated the A. fumigatus keratitis inflammatory response by inhibiting the growth of A. fumigatus, reducing the recruitment of the neutrophils and the macrophages, and inhibiting the LOX-1/ IL-1β signaling.

黄芩素在烟曲霉菌性角膜炎中的抗炎作用

黄芩素在烟曲霉菌性角膜炎中的抗炎作用

Anti-inflammatory mechanism of emodin in rats with Aspergillus fumigatus keratitis

Anti-inflammatory mechanism of emodin in rats with Aspergillus fumigatus keratitis