Schaftoside reduces inflammation in Aspergillus fumigatus keratitis through the inhibition of the TLR4/MyD88 pathway.

PURPOSE: The purpose of this research study was to investigate the impact of schaftoside on Aspergillus fumigatus (A. fumigatus) keratitis and elucidate its underlying mechanisms. METHODS: In order to establish safe experimental concentrations of schaftoside in human corneal epithelial cells (HCECs), RAW264.7 cells, and mouse models, various techniques were employed including cytotoxicity assay (CCK-8) assay, cell scratch assay, and Draize test. The therapeutic effect of schaftoside was assessed using slit-lamp biomicroscopy, clinical scores, as well as determination of neutrophil infiltration through hematoxylin and eosin (HE) staining, immunofluorescence (IF) staining, and myeloperoxidase (MPO) assay. The levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), pro-inflammatory mediators interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6 were determined using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and IF techniques. RESULTS: Schaftoside at a concentration of 160 µM displayed no harmful side effects on HCECs, RAW cells, and mouse corneas, rendering it suitable for further experiments. In a murine fungal keratitis model, schaftoside mitigated the severity of fungal keratitis by inhibiting neutrophil infiltration and reducing MPO activity. Both in vitro and in vivo experiments demonstrated that schaftoside treatment suppressed the upregulation of IL-1ß, TNF-α, and IL-6 expression, while also downregulating the expressions of TLR4 as well as MyD88 at both mRNA and protein levels. CONCLUSIONS: Schaftoside demonstrated a protective effect against A. fumigatus keratitis by reducing corneal damage through inhibition of neutrophil recruitment and downstream inflammatory cytokines. The anti-inflammatory properties of schaftoside in A. fumigatus keratitis may involve modulation of the TLR4/MyD88 pathway.

Therapeutic effect of Atractylenolide I on Aspergillus fumigatus keratitis by affecting MyD88/ NF-κB pathway and IL-1ß, IL-10 expression.

PURPOSE: Atractylenolide I (AT-I) is a natural sesquiterpene with anti-inflammatory effects. The purpose of this study was to research the anti-inflammatory effect of AT-I on Aspergillus fumigatus(A. fumigatus) keratitis in mice. METHODS: Cytotoxicity test and cell scratch test were used to determine the therapeutic concentrations of corneal infections. In vivo and in vitro studies, mouse cornea and human corneal epithelial cells (HCECs) infected with A. fumigatus were treated with AT-I or dimethyl sulfoxide (DMSO). Then, to analyze the effect of AT-I on inflammatory response, namely neutrophil or macrophage recruitment and the expression of cytokines involving MyD88, NF-κB, interleukin 1ß (IL-1ß) and interleukin 10 (IL-10). To study the effects of the drug, the techniques used include slit-lamp photography, immunofluorescence, myeloperoxidase (MPO) detection, quantitative real-time polymerase chain reaction (QRT-PCR), and western blot. At the same time, in order to explore the combined effect of the drug and natamycin, slit-lamp photographs and clinical scores were used to visually display the disease process. RESULTS: No cytotoxicity was observed under the action of AT-I at a concentration of 800 µM. In mouse models, AT-I significantly suppressed inflammatory responses, reduced neutrophil and macrophage recruitment, and decreased myeloperoxidase levels early in infection. Studies have shown that AT-I may reduce the levels of IL-1ß and IL-10 by inhibiting the MyD88/ NF-κB pathway. The drug combined with natamycin can increase corneal transparency in infected mice. CONCLUSION: AT-I may inhibit MyD88 / NF-κB pathway and the secretion of inflammatory factors IL-1 ß and IL-10 to achieve the therapeutic effect of fungal keratitis.

GSDMD, an executor of pyroptosis, is involved in IL-1ß 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-1ß 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-1ß 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-1ß 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.

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

Neutrophils are an important source of IL-1ß 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-1ß to the bioactive 17-kDa form. In the current study, we examined the role of IL-1ß in response to corneal infection with the filamentous fungus Aspergillus fumigatus and found that neutrophils were the predominant source of bioactive IL-1ß in the cornea. We also observed that caspase-11-/- mice exhibit the same susceptibility phenotype as IL-1ß-/-, 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-1ß 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-1ß 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-1ß production by neutrophils in response to pathogenic fungi.

Osteopontin contributes to effective neutrophil recruitment, IL-1ß production and apoptosis in Aspergillus fumigatus keratitis.

Fungal keratitis is a major cause of corneal ulcers, resulting in significant visual impairment and blindness. A phosphorylated glycoprotein secreted by immunocompetent cells, osteopontin (OPN) mediates cluster formation of the host fungal receptors and enhances the phagocytosis and clearance of pathogenic fungi. However, whether OPN production and function occurs in fungal keratitis is unknown. OPN expression in Aspergillus fumigatus keratitis patient corneas was assessed by quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence. Human neutrophils, THP-1 macrophages and corneal epithelial cells (HCECs) stimulated with A. fumigatus were utilized for in vitro experiments. Mouse models of A. fumigatus keratitis were developed by intrastromal injection for in vivo experiments. Using siRNAs, neutralizing antibodies, recombinant proteins and inhibitors, the production and role of OPN in A. fumigatus infection was assessed by clinical evaluation, qRT-PCR, immunofluorescence, western blotting and bioluminescence image acquisition. We observed increased corneal OPN expression in A. fumigatus keratitis patients and mouse models compared to controls. OPN production in response to A. fumigatus infection was dependent on LOX-1 and Erk1/2. Compared to controls, OPN knockdown impaired proinflammatory cytokine IL-1ß production, which was dependent on 4E-BP1. OPN knockdown decreased myeloperoxidase levels, and resulted in decreased neutrophil recruitment, higher fungal load and increased apoptosis in mouse A. fumigatus keratitis. Our results indicate that OPN is a critical component of the antifungal immune response and is essential for effective neutrophil recruitment, inflammatory cytokine production and apoptosis in A. fumigatus keratitis.

Role of PTX3 in corneal epithelial innate immunity against Aspergillus fumigatus infection.

Pentraxin3 (PTX3), a member of long pentraxin family, plays a non-redundant role in human humoral innate immunity. However, whether PTX3 is expressed by corneal epithelial cells and its role during corneal fungi infection has not yet been investigated. To identify the presence of PTX3 in cornea, the possible mechanisms involved in its expression, and also the effects on corneal anti-fungi innate immune response, clinic human corneal tissues and cultured human corneal epithelial cells (HCECs) were resorted. PTX3 mRNA and protein were detected in corneal samples and cultured HCECs, which was significantly up-regulated after exposing to Aspergillus fumigatus (A. fumigatus). Pretreated with specific inhibitors, only Syk contributed to the regulation of PTX3 expression in Dectin-1/Syk signal axis. Furthermore, among the MAPK members (p38 MAPK, ERK1/2 and JNK), only ERK1/2 and JNK were responsible for A. fumigatus induced PTX3 production. Blocking of endogenous PTX3 by siRNA down-regulated the production of IL-1ß at both mRNA and protein levels. Meanwhile, blocking of PTX3 also inhibited the phosphorylation of ERK1/2 and JNK, but not p38 MAPK. These findings demonstrate that PTX3 is expressed in human corneal epithelial cells and Syk, ERK1/2, JNK signaling pathways play an important role in the regulation of PTX3 induction. PTX3 plays a proinflammatory role in corneal epithelial anti-fungi immune response by affecting the production of IL-1ß and activation of some proinflammatory signaling pathways (ERK1/2 and JNK).

A retrospective analysis of eleven cases of invasive rhino-orbito-cerebral mucormycosis presented with orbital apex syndrome initially.

BACKGROUND: Rhino-orbito-cerebral mucormycosis(ROCM) is an invasive fungal infection that usually occurs in immunocompromised patients and sometimes presents as orbital apex syndrome(OAS) initially. It is rapidly fatal without an early diagnosis and treatment. We report the cases of invasive ROCM presenting with OAS initially in order to raise the attention of clinicians. METHODS: We retrospectively investigated eleven cases of invasive ROCM presenting initially with OAS admitted between January 2006 and December 2013. We analyzed clinical features, results of laboratory and radiological examinations, nasal endoscopy, aggressive surgical excision and debridement, and medical management outcomes of each case. RESULTS: A total of eleven cases of invasive ROCM with OAS as an initial sign were presented. Mucormycosis was accompanied by type II diabetes mellitus in nine cases, renal transplant in one case, and injury caused by traffic accident in one case. Anterior rhinoscopy revealed palatine or nasal necrotic lesions in all patients, and transethmoidal optic nerve decompression was carried out in three patients at the same time. CT scan revealed rhino-orbital-cerebral involvement in every patient. All patients were given intravenous amphotericin B. Nine patients underwent surgical debridement of necrotic tissue. Three patients survived. CONCLUSIONS: ROCM is a severe, emergent and fatal infection requiring multidisciplinary management. It may often present with OAS initially. For ophthalmologist, mucormycosis must be considered in immunocompromised patients presenting with OAS initially, and anterior rhinoscopy is imperative before hormonotherapy, even in the cases absent of ketoacidosis induced by diabetes mellitus.

Association of Dectin-1 and DC-SIGN gene single nucleotide polymorphisms with fungal keratitis in the northern Han Chinese population.

PURPOSE: Dendritic cell-associated C-type lectin-1 (Dectin-1) and dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) play a crucial role in the early procedure of fungal pathogen defenses. The present study evaluated the associations between Dectin-1 and DC-SIGN gene single nucleotide polymorphisms (SNPs) and susceptibility to fungal keratitis (FK) in the northern Han Chinese population. METHODS: The polymorphisms of Dectin-1 (rs17206002, rs3901533, rs11053613, and rs3901532) and DC-SIGN (rs4804803, rs2287886, rs735239, and rs735240) for 109 FK patients and 220 matched healthy controls were determined by PCR and DNA direct sequencing assay. RESULTS: Each SNP was consistent with Hardy-Weinberg equilibrium (p>0.05). The frequencies of genotypes and alleles for rs735239 and rs735240 (DC-SIGN) showed statistical differences between patients and control groups (p<0.05). The wild G allele of rs735239 and the wild A allele of rs735240 were significantly higher in patients (p=0.003, OR=1.766, 95% confidence interval [CI] 1.207-2.585; p=0.014, OR=1.609, 95% CI 1.100-2.355, respectively). No association with a risk of FK was found for the remaining SNPs (p>0.05) even after ruling out clinical characteristics, such as severity degree and case history. Carriers of the haplotype TC (rs4804803 and rs2287886) had a higher risk of developing fungal keratitis (p=0.007, OR=1.710, 95% CI 1.154-2.534). The distribution of haplotypes AG and GA (rs735239 and rs735240) between the two groups also showed significant differences (p=0.014, p=0.003, respectively). CONCLUSIONS: Two SNPs of DC-SIGN (rs735239 and rs735240) are associated with susceptibility to FK in the northern Han Chinese population. The haplotypes of DC-SIGN may be susceptible to the risk of FK, whereas the analysis of Dectin-1 gene polymorphisms showed no significant association with FK risk. Further research with a larger sample is recommended.

The role of Syk signaling in antifungal innate immunity of human corneal epithelial cells.

BACKGROUND: Fungal keratitis is a kind of intractable and sight-threatening diseases. Spleen-tyrosine kinase (Syk) is a non-receptor tyrosine kinase, which plays an important role in the signaling pathway of the receptors. In the current study, we investigate the expression and function of Syk in human corneal epithelial cells with Aspergillus fumigatus (A. fumigatus) infection. METHODS: Cultured telomerase-immortalized human corneal epithelial cells (THCEs) were treated with A. fumigatus hyphae with or without treatment of Syk inhibitors. Activation of Syk and the role of Syk in regulating inflammatory cytokines and chemokines expression were evaluated. The mRNA expression was determined by real time PCR, and protein activation was measured by western blotting. RESULTS: Syk protein was detected in THCEs, and its activation was enhanced after treatment of A. fumigatus hyphae. Expression of inflammatory cytokines (IL-1ß and IL-6) and chemokines (IL-8 and CXCL1) mRNA were significantly increased after stimulation of A. fumigatus hyphae in THCEs. Activation of Syk and expression of IL-1ß, IL-6, IL-8 and CXCL1 by A. fumigatus hyphae were blocked by Syk inhibitors. CONCLUSION: These findings demonstrate that normal human corneal epithelial cells produce Syk, and Syk activation plays an important role in regulating A. fumigatus hyphae-induced inflammatory responses in THCEs.

CD3ε of a pan T cell marker involved in mouse Aspergillus fumigatus keratitis.

AIM: To explore whether CD3ε is involved in the adaptive immunity of Aspergillus fumigatus (A. fumigatus) keratitis in mice and the role of innate and adaptive immunity in it. METHODS: Mice models of A. fumigatus keratitis were established by intra-stromal injection and corneal epithelial scratching. Subconjunctival injections of natamycin, wedelolactone, LOX-1 inhibitor (poly I) or Dectin-1 inhibitor (laminarin) were used to treat mice with A. fumigatus keratitis. Mice were pretreated by intraperitoneal injection of anti-mouse CD3ε. We observed the corneal infection of mice under the slit lamp microscope and made a clinical score. The protein expression of CD3ε and interleukin-10 (IL-10) was determined by Western blotting. RESULTS: With the disease progresses, the degree of corneal opacity and edema augmented. In the intra-stromal injection models, CD3ε protein expression began to increase significantly on the 2nd day. However, in the scraping epithelial method models, CD3ε only began to increase on the 3rd day. After natamycin treatment, the degree of corneal inflammation in mice was significantly attenuated on the 3rd day. After wedelolactone treatment, the severity of keratitis worsened. And the amount of CD3ε protein was also reduced, compared with the control group. By inhibiting LOX-1 and Dectin-1, there was no significant difference in CD3ε production compared with the control group. After inhibiting CD3ε, corneal ulcer area and clinical score increased, and IL-10 expression was downregulated. CONCLUSION: As a pan T cell marker, CD3ε participate in the adaptive immunity of A. fumigatus keratitis in mice. In our mice models, the corneas will enter the adaptive immune stage faster. By regulating IL-10, CD3ε exerts anti-inflammatory and repairs effects in the adaptive immune stage.

Thymol Ameliorates Aspergillus fumigatus Keratitis by Downregulating the TLR4/ MyD88/ NF-kB/ IL-1ß Signal Expression and Reducing Necroptosis and Pyroptosis.

Fungal keratitis is a refractory kind of keratopathy. We attempted to investigate the anti-inflammatory role of thymol on Aspergillus fumigatus (A. fumigatus) keratitis. Wound healing and fluorescein staining of the cornea were applied to verify thymol's safety. Mice models of A. fumigatus keratitis underwent subconjunctival injection of thymol. The anti-inflammatory roles of thymol were verified by hematoxylin-eosin (HE) staining, slit lamp observation, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. In contrast with the DMSO group, more transparent corneas and less inflammatory cells infiltration were detected in mice treated with 50 µg/ml thymol. Thymol downregulated the synthesis of TLR4, MyD88, NF-kB, IL-1ß, NLRP3, caspase 1, caspase 8, GSDMD, RIPK3 and MLKL. In summary, we proved that thymol played a protective part in A. fumigatus keratitis by cutting down inflammatory cells aggregation, downregulating the TLR4/ MyD88/ NF-kB/ IL-1ß signal expression and reducing necroptosis and pyroptosis.

Disulfiram inhibits IL-1ß secretion and inflammatory cells recruitment in Aspergillus fumigatus keratitis.

PURPOSE: Disulfiram, an inhibitor of gasdermin D-induced pore formation, is known to suppress interleukin (IL)-1ß secretion and pyroptosis. However, its effects on fungal keratitis remain unknown. Therefore, we investigated the role of disulfiram in Aspergillus fumigatus keratitis. METHODS: In vitro, Cell Count Kit-8 (CCK8) assay and cell scratch test were performed to determine optimal concentration. In vivo and in vitro experiments were conducted in a mouse model, human neutrophils, and mouse peritoneal macrophages. We pre-treated the mice or cells with disulfiram and infected them with A. fumigatus at specific times. We subsequently evaluated the development of fungal keratitis lesions, the recruitment of inflammatory cells, and the production of inflammatory cytokines using slit lamp microscopy, clinical evaluation, quantitative reverse transcription polymerase chain reaction, immunofluorescence staining, enzyme-linked immunosorbent assay, and western blotting. We also used slit lamp microscopy and clinical evaluation to assess the effect of natamycin with or without disulfiram. RESULTS: Disulfiram at 20 µM has no significant cytotoxic effect and does not affect cell migration. In the mouse model, disulfiram significantly suppressed inflammatory responses, reduced neutrophil and macrophage recruitment, and down-regulated myeloperoxidase and nitric oxide synthase levels at earlier stages of infection. Disulfiram had no effect on IL-1ß production and maturation, but it inhibited IL-1ß secretion in macrophages. Disulfiram combined with natamycin significantly increased corneal transparency in the mice model. CONCLUSION: Overall, disulfiram reduced the host immune response in fungal keratitis by attenuating neutrophil and macrophage recruitment and inhibiting IL-1ß secretion in macrophages. Disulfiram in combination with antifungal agents may serve as a novel therapeutic method for reducing corneal opacity in fungal keratitis.

Caspase-8 is involved in pyroptosis, necroptosis and the maturation and release of IL-1ß in Aspergillus fumigatus keratitis.

PURPOSE: To explore the role of caspase-8 in mediating the transition between different death modes in fungal keratitis. METHODS: The expression of caspase-8 in Aspergillus fumigatus (A. fumigatus) keratitis was detected using western blotting and immunofluorescence. After subconjunctival injection of Z-IETD-FMK (caspase-8 inhibitor) or VX765 (caspase-1 inhibitor), the mice corneas of A. fumigatus keratitis were observed and scored under a slit lamp. Colony plate count, immunofluorescence staining, western blotting and qRT-PCR experiments were used to detect fungal load, inflammatory cells, and the production of related mRNAs and proteins. In vitro experiments, the LDH release test, Cell Count Kit-8(CCK-8) assay, ELISA, qRT-PCR and western blotting were used to detect cell viability, related mRNAs and proteins. RESULTS: The caspase-8 protein was upregulated following fungal infection. Compared with the A. fumigatus keratitis group, the mice treated with Z-IETD-FMK had heavier corneal turbidity, higher clinical scores, more fungal load and fewer inflammatory cells. The expression of NLRP3, cleaved-caspase-1, N-GSDMD, and IL-1ß in the fungal infection group after Z-IETD-FMK pretreatment were downregulated, while RIPK3 and p-MLKL were upregulated. In the fungal infection group after VX765 pretreatment, the expression of cleaved-caspase-8 was up-regulated, while N-GSDMD was downregulated. CONCLUSIONS: Caspase-8 is involved in the early immune defense response of A. fumigatus keratitis. It is essential for the recruitment of inflammatory cells and the clearance of the fungus. In A. fumigatus keratitis, activated caspase-8 promoted the caspase-1/GSDMD signaling pathway to participate in pyroptosis, inhibited RIPK3/MLKL signaling pathway-mediated necroptosis, and promoted IL-1ß maturation and release by activating the NLRP3 inflammasomes.

CCPG1 involved in corneal Aspergillus fumigatus infection.

AIM: To investigate whether non-canonical autophagy transport receptor cell cycle progression 1 (CCPG1) is involved in the corneal antifungal immune response. METHODS: Human corneal epithelial cells (HCECs) and human myeloid leukemia mononuclear cells (THP-1) macrophages stimulated by Aspergillus fumigatus (A. fumigatus) were used as cell models. The expression of CCPG1 mRNA was detected by qRT-PCR. Western blot was used to determine the protein expression of CCPG1 and interleukin-1ß (IL-1ß). The dectin-1 neutralizing antibody was used to detect the association between dectin-1 and CCPG1. Immunofluorescence was used to observe the colocalization of CCPG1 and C-type lectin-like receptor-1 (CLEC-1) in THP-1 macrophages. RESULTS: The expression of CCPG1 started to increase at 4h after infection and increased in a time-dependent manner in HCECs and THP-1 macrophages. With dectin-1 neutralizing antibody pretreatment, the expression of IL-1ß was down-regulated. CCPG1 up-regulation in response to A. fumigatus infection was independent of dectin-1. Immunofluorescence showed the colocalization of CCPG1 and CLEC-1 in THP-1 macrophages. CONCLUSION: As a specific autophagy protein of non-canonical autophagy pathway, CCPG1 is involved in corneal infection with A. fumigatus.

Thymol Protects against Aspergillus Fumigatus Keratitis by Inhibiting the LOX-1/IL-1ß Signaling Pathway.

OBJECTIVE: To explore the anti-inflammatory effects and mechanisms of action of thymol in Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: The minimum inhibitory concentration of thymol against A. fumigatus was detected. To characterize the anti-inflammatory effects of thymol, mouse corneas and human corneal epithelial cells were pretreated with thymol or dimethyl sulfoxide (DMSO) before infection with A. fumigatus spores. Slit-lamp microscopy, immunohistochemistry, myeloperoxidase detection, quantitative real-time polymerase chain reaction, and Western blotting were used to assess infection. Neutrophil and macrophage recruitment, in addition to the secretion of LOX-1 and IL-1ß, were quantified to evaluate the relative contribution of thymol to the inflammatory response. RESULTS: We confirmed that the growth of A. fumigatus was directly inhibited by thymol. In contrast with the DMSO group, there was a lower degree of inflammation in the mouse corneas of the thymol-pretreated group. This was characterized by significantly lower clinical scores, less inflammatory cell infiltration, and lower expression of LOX-1 and IL-1ß. Similarly, in vitro experiments indicated that the production of LOX-1 and IL-1ß was significantly inhibited after thymol treatment, in contrast with the DMSO-pretreated group. CONCLUSION: Our findings demonstrate that thymol exerted a direct fungistatic activity on A. fumigatus. Furthermore, thymol played a protective role in fungal keratitis by inhibiting LOX-1/IL-1ß signaling pathway and reducing the recruitment of neutrophils and macrophages.

[Expression of substance P in experimental fungal keratitis].

OBJECTIVE: To detect the change process of substance P (SP)expression and the difference between fungus and bacterial keratitis and explore effects of SP on the damage and repair process of keratitis model of Wistar rats. METHODS: Wistar rats were divided randomly as blank controlled group, fungal keratitis group and bacterial keratitis group. Inoculate fusarium and staphylococcus into cornea of Wistar rats to make animal keratitis model. Choose the right eye as experimental eye and the left eye as control. After the model was created successfully, 25 rats were executed randomly at 1, 5, 8, 10 and 14 days after the surgery. Detailed record and hematoxylin-eosin dye were done to each group to examine the process of ulcer. Expression of SP were detected through immunohistochemistry and reverse transcription polymerase chain reaction. Use variance analysis, T test and Q test to evaluate the results. RESULTS: One day after infection, the cornea of experimental group showed ulcer with disordered shallow matrix layer collagenous fibre arrangement and neutral granular cell infiltration; after 5 days, ulcer worsened and granular cells infiltrated through the whole layer; after 8 days, ulcer shrinked and fibroblast began to increase with new vessels formed; after 10 days, new vessels began to decrease; after 14 days, the matrix level textile fiber assumes scar type restructuring. The expression of SP increased in endothelium cells, inflammatory cells, fibroblast cells and endothelium of new vessels 1 d after surgery (absorbance for fusarium and staphylococcus group were 0.3313 ± 0.0133 and 0.3995 ± 0.0191 respectively; mRNA were 0.4525 ± 0.0170 and 0.5532 ± 0.0258), peaked at 8 d (for fusarium and staphylococcus group were 0.5525 ± 0.0171 and 0.7050 ± 0.0119 respectively; mRNA were 0.5975 ± 0.0221 and 0.7150 ± 0.0238), began to decrease at 10 d (for fusarium and staphylococcus group were 0.1533 ± 0.0176 and 0.3125 ± 0.0170 respectively; mRNA were 0.2416 ± 0.0082 and 0.4835 ± 0.0082) and dropped to lower than normal at 14 d (for fusarium and staphylococcus group were 0.1150 ± 0.0128 and 0.1675 ± 0.0126 respectively; mRNA were 0.1275 ± 0.0126 and 0.2325 ± 0.0171), which had significant difference (the absorbance of SP: for fusarium group, F = 832.24, q = 35.3675, 12.9044, 27.3621, 34.6506, 22.4632, 62.7296, 70.0182, 40.2664, 47.5550, 7.2886, P < 0.01;for staphylococcus group F = 636.17, q = 38.7494, 11.4245, 10.8298, 28.6082, 27.3249, 49.5791, 67.3575, 22.2543, 40.0327, 17.7784, P < 0.01. mRNA expression: for fusarium group F = 658.60, q = 18.9941, 9.5132, 27.4719, 42.0007, 9.4809, 46.4661, 60.9948, 36.9852, 51.5139, 14.5287, P < 0.01; for staphylococcus group F = 335.13, q = 16.9266, 4.1677, 7.1081, 32.4724, 12.7589, 24.0347, 49.3990, 11.2759, 36.6402, 25.3643, P < 0.01). Two experimental groups showed similar changes as time changes, the bacterial group showed more SP expression than fungal group, which had significant difference (absorbance of SP t = 6.5493, 7.3867, 16.0505, 14.5479, 6.5360, P < 0.05; mRNA expression t = 7.2878, 9.5232, 8.8149, 43.6256, 11.1269, P < 0.05). Controlled eyes showed increased SP expression at 1 d (absorbance was 0.2840 ± 0.0212; mRNA was 0.3950 ± 0.0129) and dropped to normal at 5 d (absorbance was 0.2125 ± 0.0174; mRNA was 0.3321 ± 0.0041). There was significant difference between bacterial group, fungal group and controlled eyes (absorbance of SP: compared with fusarium group t = 4.2261, 18.3314, 35.5163, 5.3609, 13.4826; compared with staphylococcus group t = 9.0508, 25.6639, 63.4924, 7.4828, 7.1301, P < 0.05. mRNA expression: compared with fusarium group t = 6.0249, 31.9158, 26.0413, 9.1550, 19.1741; compared with staphylococcus group t = 12.2636, 53.4404, 36.8727, 15.8687, 8.2939, P < 0.05). CONCLUSION: SP possibly participated in the early time damage and the later period repair process in fungus keratitis and its lower expression level possibly participated in the mechanism of lighter ache in fungus keratitis.

CLEC-1 Acts as a Negative Regulator of Dectin-1 Induced Host Inflammatory Response Signature in Aspergillus fumigatus Keratitis.

Purpose: C-type lectin-like receptor-1 (CLEC-1) is a member of the Dectin-1 cluster of pattern recognition receptors (PRRs). It is involved in host immunity, has immunoregulatory function, and supports allograft tolerance. Our study aimed to describe the role of CLEC-1 in response to fungal keratitis, in situ, in vivo, and in vitro. Methods: Quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence were used to detect the expression of CLEC-1 in corneas of patients with Aspergillus fumigatus (A. fumigatus) keratitis. In vitro and in vivo experiments were designed in THP-1 macrophages and C57BL/6 mouse models, respectively. The expression of CLEC-1 in corneas of mice model was determined by qRT-PCR, Western blot, and immunofluorescence. CLEC-1 overexpression in mouse corneas was achieved by intrastromal injection of adeno-associated virus (AAV) vectors. Disease response was evaluated by slit-lamp photography, clinical score, and colony forming unit (CFU). Bioluminescence imaging system image acquisition, myeloperoxidase (MPO) assays, immunofluorescence staining, qRT-PCR, and Western blot were used to investigate the role of CLEC-1. To further define the role of CLEC-1, we used lentivirus vectors to overexpress CLEC-1 or/and Dectin-1 in THP-1 macrophages. Results: The expression of CLEC-1 was increased in corneas of patients with A. fumigatus keratitis. In corneas of mice from the A. fumigatus keratitis model, the expression of CLEC-1 was decreased in the acute inflammatory stage and increased during convalescence. Following Natamycin treatment, CLEC-1 was upregulated in A. fumigatus keratitis mice. Compared with normal C57BL/6 mice, overexpression of CLEC-1 converted the characteristic susceptible response to resistance, as demonstrated by slit-lamp photography and clinical score. In vivo studies revealed decreased MPO levels and neutrophils recruitment and higher fungal load after the upregulation of CLEC-1. Compared with control corneas, CLEC-1 overexpression impaired corneal pro-inflammatory cytokine IL-1ß production. Conclusions: These findings demonstrate that CLEC-1 may act as a negative regulator of Dectin-1 induced host inflammatory response via suppressing neutrophils recruitment and production of pro-inflammatory cytokine IL-1ß production in response to A. fumigatus keratitis.

SARS­CoV­2 spike protein­induced host inflammatory response signature in human corneal epithelial cells.

Coronavirus disease 2019 (COVID­19), caused by the severe acute respiratory syndrome coronavirus­2 (SARS­CoV­2), led to an outbreak of viral pneumonia in December 2019. The present study aimed to investigate the host inflammatory response signature­caused by SARS­CoV­2 in human corneal epithelial cells (HCECs). The expression level of angiotensin­converting enzyme 2 (ACE2) in the human cornea was determined via immunofluorescence. In vitro experiments were performed in HCECs stimulated with the SARS­CoV­2 spike protein. Moreover, the expression levels of ACE2, IL­8, TNF­α, IL­6, gasdermin D (GSDMD) and IL­1ß in HCECs were detected using reverse transcription­quantitative PCR and/or western blotting. It was identified that ACE2 was expressed in normal human corneal epithelium and HCECs cultured in vitro. Furthermore, the expression levels of IL­8, TNF­α and IL­6 in HCECs were decreased following SARS­CoV­2 spike protein stimulation, while the expression levels of GSDMD and IL­1ß were increased. In conclusion, the present results demonstrated that the SARS­CoV­2 spike protein suppressed the host inflammatory response and induced pyroptosis in HCECs. Therefore, blocking the ACE2 receptor in HCECs may reduce the infection rate of COVID­19.

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

PURPOSE: Nerolidol, 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. METHODS: The 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 1ß (IL-1ß). Techniques used were the slit lamp, immunofluorescence, myeloperoxidase (MPO) detection, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: Nerolidol 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-1ß were reduced compared with the PBS group. Additionally, in vitro studies showed that treatment with nerolidol inhibited the production of the LOX-1 / IL-1ß levels in A. fumigatus stimulated HCECs. CONCLUSION: Nerolidol 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-1ß signaling.

The expression of lacrimal androgen-binding proteins in mice Pseudomonas aeruginosa keratitis.

AIM: To investigate the expression of lacrimal androgen-binding proteins (ABPs) in mice Pseudomonas aeruginosa (P. aeruginosa) keratitis. METHODS: P. aeruginosa mice model from different gender was developed by intra-stromal injection. The expression of lacrimal ABPs in lacrimal gland specimens from P. aeruginosa keratitis mice was detected by the quantitative polymerase chain reaction (qRT-PCR). Corneal virulence was evaluated based on clinical scores. To study the mechanism of lacrimal ABPs' expression, experimental subjects were pre-treated with 4E-BP1 inhibitor, and were used to evaluate the expression levels by qRT-PCR. RESULTS: Compared with control groups, the expression of ABPα, ABPη and ABPζ in lacrimal gland from P. aeruginosa keratitis mice had no meaningful changes, while ABPε and ABPδ were significantly higher at 1d after infection. The expression of ABPδ in lacrimal gland of male mice was higher than female mice, regardless of whether or not P. aeruginosa keratitis occurred. After 4E-BP1 inhibitor subconjunctival injection or lacrimal injection, the expression of ABPδ and ABPε has no significant change compared with the control group. CONCLUSION: ABPδ and ABPε secreted by mice lacrimal gland may involve in the progress of alleviating the severity of corneal damage in P. aeruginosa keratitis. The expression of ABPδ and ABPε upon P. aeruginosa infection is independent of cap-dependent mRNA translation activated by 4E-BP1.