Identifying genetic susceptibility to Aspergillus fumigatus infection using collaborative cross mice and RNA-Seq approach

Background:Aspergillus fumigatus(Af)is one of the most ubiquitous fungi and its infection potency is suggested to be strongly controlled by the host genetic back-ground.The aim of this study was to search for candidate genes associated with host susceptibility to Aspergillus fumigatus(Af)using an RNAseq approach in CC lines and hepatic gene expression.Methods:We studied 31 male mice from 25 CC lines at 8 weeks old;the mice were infected with Af.Liver tissues were extracted from these mice 5 days post-infection,and next-generation RNA-sequencing(RNAseq)was performed.The GENE-E analysis platform was used to generate a clustered heat map matrix.Results:Significant variation in body weight changes between CC lines was ob-served.Hepatic gene expression revealed 12 top prioritized candidate genes differ-entially expressed in resistant versus susceptible mice based on body weight changes.Interestingly,three candidate genes are located within genomic intervals of the previ-ously mapped quantitative trait loci(QTL),including Gm16270 and Stox1 on chromo-some 10 and Gm11033 on chromosome 8.Conclusions:Our findings emphasize the CC mouse model's power in fine mapping the genetic components underlying susceptibility towards Af.As a next step,eQTL analysis will be performed for our RNA-Seq data.Suggested candidate genes from our study will be further assessed with a human cohort with aspergillosis.

北部湾海洋真菌Aspergillus fumigatus DL-p0m-g2的化学成分及药理活性研究

为获得具有生物活性的化合物,对一株分离于北部湾钉螺菌株Aspergillus fumigatus DL-p0m-g2的化学成分及药理活性进行研究。综合应用反相ODS(octadecylsilyl)柱色谱、半制备液相等多种色谱学方法进行分离纯化,根据化合物理化性质、质谱和核磁共振波谱学数据进行结构鉴定,并对分离得到的化合物进行细胞毒活性、抑菌活性和胆固醇转运蛋白NPC1L1(NPC1-like intracellular cholesterol transporter 1)蛋白结合等生物活性评价。实验共分离得到21个生物碱类化合物和1个甾体,分别鉴定为6-methoxyspirotryprostatin B(1)、spirotryprostatin A(2)、fumitremorgin C(3)、cyclotryprostatin A(4)、fumitremorgin B(5)、pseurotin A(6)、azaspirofuran A(7)、azaspirofuran B(8)、cephalimysin C(9)、cephalimysin B(10)、fumiquinazoline C(11)、fumiquinazoline B(12)、fumiquinazoline A(13)、fumiquinazoline D(14)、fumiquinazoline F(15)、tryprostatin B(16)、verruculogen(17)、chaetominine(18)、bisdethiobis(methylthio)glitoxin(19)、helvolic acid(20)、7-deacetylpyripyropene A(21)、terezine D(22)。其中化合物6对人肝癌细胞(Hep G2)、人肺癌细胞(A549)、人直肠癌细胞(HCT116)有一定的细胞毒活性;化合物1、3和20对金黄色葡萄球菌表现出抑菌活性;化合物14与NPC1L1蛋白具有较好的结合,显示其在降脂药物开发中的研究潜力。

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 intrastromal injection models,CD3εprotein expression began to increase significantly on the 2^(nd) day.However,in the scraping epithelial method models,CD3ε only began to increase on the 3^(rd) day.After natamycin treatment,the degree of corneal inflammation in mice was significantly attenuated on the 3^(rd) 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 antiinflammatory and repairs effects in the adaptive immune stage.

水母共附生真菌Aspergillus fumigatus SCSIO41214中生物碱类化合物研究

从水母共附生真菌Aspergillus fumigatus SCSIO41214中分离得到8个已知生物碱类化合物。这些化合物主要采用包括正相硅胶、反相硅胶等常规柱层析技术,以及高效液相色谱进行分离纯化。首次通过核磁共振波谱等多种现代波谱技术确定了化合物1的平面结构,通过单晶衍射技术确定了其相对构型,化合物2-8是通过波谱分析并结合文献数据比对确定了化合物的结构。体外抗炎活性研究筛选发现,化合物4在10μmol·L^(-1)浓度下显示弱的抑制RAW264.7细胞中LPS引起的NO的释放。

越前水母共附生真菌Aspergillus fumigatus sp.次级代谢产物研究

采用硅胶柱层析、Sephadex LH-20凝胶柱层析、中压反相柱层析以及半制备高效液相色谱等方法对韩国南部海岸来源的越前水母共附生真菌A.fumigatus的大米发酵产物进行分离纯化,并利用核磁共振、质谱等波谱学方法结合其理化性质及文献数据鉴定了7个单体化合物的结构:sinulariapeptide C(1)、simplicilliumtide A(2)、simplicilliumtide B(3)、sinulariapeptide D(4)、4-羟基-17 R-甲基降甾醇(5)、3-羟基苯乙酸甲酯(6)、邻苯二甲酸二丁酯(7)。化合物1~7均为首次从越前水母来源的菌属中分离得到。

The roles of surfactant protein D during Aspergillus fumigatus infection in human corneal epithelial cells

AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial (HCE) cells Exposed to aspergillus fumigatus (AF) antigens. METHODS: HCE cells cultured 47 in vitro with AF antigens and sampled at 0, 0.5, 1 hour, 2, 4, 6 and 8 hours. The Expression of SP-D mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of SP-D protein was shown by ELISA and immunocytochemistry SP methods. The expression of NF-kappa B and relative downstream cytokines such as TNF-alpha, IL-1 beta, IL-8 and IL-10 in supernatant fluid were measured by ELISA. RESULTS: SP-D mRNA and protein were detected in untreated HCE cells. The expression of SP-D and the relative downstream cytokines rose after being stimulated with AF antigens. SP-D mRNA began to rise at 0.5 hour and the most significantly peak was in 2 hours. The protein of SP-D in supernatant fluid had the same trend with mRNA. Immunocytochemistry of SP-D showed positive expression and gradually increased to 6 hours, and then the expression began to decline. NF-kappa B was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-alpha, IL-1 beta, IL-8 and IL-10 began to rise after given AF antigens 1 hour and were 1.82, 1.43, 1.12 and 1.28 times higher than the untreated HCE cells separately. The expression of TNF-alpha and IL-1 beta reached the peak at 2 hours, separately 2.80 and 2.86 times than the untreated. The expression of IL-8 and IL-10 gradually increased with a time-dependent manner. ' CONCLUSION: HCE cells exists SP-D and it may play a significant role in pathogenesis of keratomycosis. AF may induce human corneal epithelial cells to express inflammatory cytokines via SP-D and NF-kappa B pathway. SP-D possibly mediates the recognition to AF mycelium.

Comparison of four supports for adsorption of reactive dyes by immobilized Aspergillus fumigatus beads

Four materials, sodium carboxymethylcellulose （Na-CMC）, sodium alginate （SA）, polyvinyl alcohol （PVA）, and chitosan （CTS）, were prepared as supports for entrapping fungus Aspergillusfumigatus. The adsorption of synthetic dyes, Reactive Brilliant Blue KN- R, and Reactive Brilliant Red K-2BP, by these immobilized gel beads and plain gel beads was evaluated. The adsorption efficiencies of Reactive Brilliant Red K-2BP and Reactive Brilliant Blue KN-R by CTS immobilized beads were 89.1% and 93.5% in 12 h, respectively. The adsorption efficiency by Na-CMC immobilized beads was slightly lower than that of mycelial pellets. But the dye culture mediums were almost completely decolorized in 48 h using the above-mentioned two immobilized beads （exceeding 95%）. The adsorption efficiency by SA immobilized beads exceeded 92% in 48 h. PVA-SA immobilized beads showed the lowest adsorption efficiency, which was 79.8% for Reactive Brilliant Red K-2BP and 92.5% for Reactive Brilliant Blue KN-R in 48 h. Comparing the adsorption efficiency by plain gel beads, Na-CMC plain gel beads ranked next to CTS ones. SA and PVA-SA plain gel beads hardly had the ability of adsorbing dyes. Subsequently, the growth of mycelia in Na-CMC and SA immobilized beads were evaluated. The biomass increased continuously in 72 h. The adsorption capacity of Reactive Brilliant Red K-2BP and Reactive Brilliant Blue KN-R by Na-CMC immobilized beads was 78.0 and 86.7 mg/g, respectively. The SEM micrographs show that the surface structure of Na-CMC immobilized bead is loose and finely porous, which facilitates diffusion of the dyes.

海南东寨港红树林内生真菌Aspergillus fumigatus SAS10吡喃酮类代谢产物

采用大米培养基,对无瓣海桑内生真菌Aspergillus fumigatus SAS10进行静置发酵,运用硅胶柱色谱、Sephadex LH-20凝胶柱色谱、以及半制备高效液相等分离手段对发酵产物进行分离纯化,获得7个化合物。经分析质谱、核磁等波谱数据,并通过与文献对比鉴定化合物为:aspergyllone(1),(R)-2-(羟基(苯基)甲基)-4H-吡喃-4-酮(2),carbonarone A(3),campyrone A(4),campyrone C(5),asperfurandione A(6)和dibutylterephthalate(7)。所得7个化合物均为首次报道为烟曲霉次级代谢产物。

Dectin-1 expression at early period of Aspergillus fumigatus infection in rat's corneal epithelium

AIM:To investigate the expression of dendritic cell-associated C-type lectin-1 (dectin-1) at the early period of Aspergillus fumigatus infection in rat’s corneal epithelium. ·METHODS:A total of 72 Wistar rats were randomly divided into three groups:A, B and C. The right eyes were chosen as experimental eyes. Group A was control group. Rats in group B were not inoculated with Aspergillus fumigatus. Group C was taken as Aspergillus fumigatus keratitis model. Rats in group B and C (six from each group) were executed randomly at 4, 8, 16 and 24 hours after experimental model being established to assess the expression of dectin-1 mRNA through real-time PCR. Another six rats in group B and C were executed randomly at 24 hours to assess the expression of dectin-1 protein through immunohistochemistry. ·RESULTS:The results of real-time PCR indicated that dectin-1 mRNA expression was low in corneal epithelium of normal rats’. There was no significantly difference of dectin-1 mRNA expression in group A and B (P 】0.05). The expression of Aspergillus fumigatus infected corneal epithelium increased gradually after 8 hours in group C. The synchronous expression of group A and C had significant difference (P 【0.01). Immunohistochemisty discovered that dectin-1 receptor existed in normal rat’s corneal epithelium . Dectin-1 protein increased after 24 hours in group C. There was a significant difference of synchronous expression in group B and C(P【0.01). · CONCLUSION:Dectin-1 exists in rat’s cornealepithelium and its expression significantly increases at the early period of Aspergillus fumigatus infection. Dectin-1 is a pattern recognition receptor that expresses in corneal epithelium and involves in immune response to Aspergillus fungal keratitis.

Nucleotide oligomerization domain 2 contributes to the innate immune response in THCE cells stimulated by Aspergillus fumigatus conidia

AIM: To investigate the expression of nucleotide oligomerization domain 2 (NOD2) in the immortalized human corneal epithelial cell line (THCE), and its role in the innate immune response triggered by inactive Aspergillus fumigatus (Af) conidia. METHODS: The normal THCE cells were investigated as controls. After incubation with inactive Af conidia for 0.5, 2, 4, 6, and 8 hours, THCE cells were harvested, mRNA expression of NOD2 and receptor interacting protein 2 (RIP2) was detected by RT-PCR. Intracellular proteins including NOD2, NF-kappa B and proinflammatory cytokines such as TNF-alpha, IL-8, IL-6 in the cell supernatant were analyzed by ELISA. RESULTS: Our data indicate that NOD2 expressed in the normal THCE cells. After triggered by the inactive Af conidia, the expression of NOD2, RIP2 mRNA and the secretion of NOD2, NF-kappa B, TNF-alpha, IL-8, IL-6 both increased in a time-depended manner, and reached the peak point at 4, 6, 6, 4, 6, 6, 4 hours, respectively. And after pretreated with NOD2 neutralizing antibody, the expression of RIP2, NF-kappa B, TNF-alpha, IL-8 both decreased dramatically at the peak point, while the secretion of IL-6 changed little. CONCLUSION: The results of this study suggest that NOD2 exists and expresses in the THCE cells, and contributes to the innate immune responses triggered by inactive Afconidia by induction of proinflammatory cytokines such as TNF-alpha and IL-8 through the NF-kappa B pathway.

Expression of indoleamine 2,3-dioxygenase in a murine model of Aspergillus fumigatus keratitis

AIM： To observe the presence and expression of indoleamine 2,3-dioxygenase（IDO） during the corneal immunity to Aspergillus fumigatus（A. fumigatus） in the murine models.·METHODS： The murine model of fungal keratitis was established by smearing with colonies of A. fumigatus after scraping central epithelium of cornea and covering with contact lenses in C57BL/6 mice. The mice were randomly divided into control group, sham group and A.fumigatus keratitis group. The cornea was monitored daily using a slit lamp and recorded disease score after infection. Corneal lesion was detected by immunofluorescence staining. IDO m RNA and protein were also detected by quantitative reverse transcription-polymerase chain reaction（q RT-PCR） and Western blot.· RESULTS： The disease score and slit lamp photography indicated that disease severity was consistent with corneal inflammation in the murine models, and the disease scores in A. fumigatus keratitis group were obviously higher than those in the sham group. By immunofluorescence staining, IDO was mainly localized in corneal epithelium and stroma in the murine corneal tissues with A. fumigatus keratitis. Compared with the sham group, IDO m RNA expression was significantly enhanced in corneal epithelium infected by A. fumigatus. Furthermore, IDO protein expression detected by Western blot was in accord with transcript levels of IDO m RNA measured by q RT-PCR. IDO protein expression was enhanced after A. fumigatus infection compared with the sham group.·CONCLUSION： IDO is detected in corneal epithelium and stroma locally, which indicates IDO takes part in the pathogenesis of A. fumigatus keratitis and plays a key role in immune regulation at the early stage.

Regulation of lipoxygenase-1 and Dectin-1 on interleukin-10 in mouse Aspergillus fumigatus keratitis

AIM： To investigate the regulation of lipoxygenase （LOX）-1 and Dectin-1 on interleukin-10 （IL-10） production in mice with Aspergillus fumigatus （A. fumigatus） keratitis. METHODS： The corneas of C57BL/6 mice were pretreated with LOX-1 inhibitor Poly（I） or Dectin-1 siRNA separately before the infection of A. fumigatus. Polymerase chain reaction （PCR） and Western blot were used to detect the expression of IL-10. RESULTS： The mRNA and protein expressions of IL-10 were significantly increased in mice with A. fumigatus keratitis. Compared with the group pretreated with sterile water before infection, Poly（I） pretreatment suppressed IL-10 expression significantly. Compared with the group pretreated with scrambled siRNA before infection, Dectin-1 siRNA pretreatment significantly reduced IL-10 expression in response to A. fumigatus infection. CONCLUSION： LOX-1 and Dectin-1 regulate IL-10 production in mouse A. fumigatus keratitis.

The role of Dectin-1/Raf-1 signal cascade in innate immune of human corneal epithelial cells against Aspergillus fumigatus infection

AIM： To investigate the expression of the v-raf-1murine leukemia viral oncogene homolog 1（Raf-1） and its role in the innate immune response of human corneal epithelial cells（HCECs） infected by Aspergillus fumigatus.METHODS： HCECs were cultured in vitro.They were randomly divided into 4 groups,including control group,Aspergillus fumigatus group,GW5074（an inhibitor of Raf-1） group and Laminarin [an inhibitor of Dendriti-cell-associated C-type lectin 1（Dectin-1）] group.The protein expression level of total Raf-1 and p-Raf-1 was measured by Western blot.The expression of IL-6 and IL-8 m RNA in each group was detected by real-time polymerase chain reaction.RESULTS： In Aspergillus fumigatus group,total Raf-1 protein levels in HCECs remained unchanged at 5,15,30 and 45min after infection,while p-Raf-1 expression was significantly enhanced at 30 min after infection compared with control group.However,the expression of p-Raf-1 was apparently declined after treated with GW5074 or Laminarin compared with Aspergillus fumigatus group.The expression levels of IL-6,IL-8 m RNA were significantly increased after stimulation with fumigatus compared with control group.Pre-treated with GW5074 significantly inhibited Aspergillus fumigatus-induced upregulation of IL-8 and IL-6.CONCLUSION： Aspergillus fumigatus stimulation can elevate the expression of p-Raf-1 in HCECs in vitro.Dectin-1/Raf-1 signal pathway may play a role on regulating the expression of inflammatory cytokines,including IL-6 and IL-8.

TREM-1 expression in rat corneal epithelium with Aspergillus fumigatus infection

AIM: To investigate the expression of triggering receptor expressed on myeloid cells-1(TREM-1) in the aberrant inflammation within the corneal epithelium at early period of fungal infection.METHODS: A total of 65 Wistar rats were randomly divided into control group, sham group and fungal keratitis(FK) group, in which the cornea was infected by Aspergillus fumigatus(A. fumigatus). After executed randomly at 8, 16, 24, 48 and 72 h after experimental model being established, the severity of keratomycosis in rats was scored visually with the aid of a dissecting microscope and slit lamp. Then corneas in three groups were collected to assess the expression of TREM-1through quantitative reverse transcription-polymerase chain reaction(RT-PCR), immunofluorescence technique and Western blot analysis. The correlation between FK inflammation and expression of TREM-1 was also analyzed.RESULTS: Corneal inflammation scores increased with time after fungal infection(F =49.74, P =0.000). The inflammation scores in FK group were obviously higher than those in sham group on the whole(F =137.78, P =0.000). Levels of TREM-1 in the infected rat corneal epithelium had elevated at 8h and peaked at 48h(P 【0.001,compared with control group). Western blot analysis also showed an obviously elevated TREM-1 level in rat corneal epithelium at 24 h and 48 h after fungal infection.Immunofluorescence technique showed that TREM-1mainly existed in corneal epithelium and infected corneal stoma of rat. TREM-1 protein expression was enhanced after fungal infection. Moreover, severity of FK inflammation was significantly related to TREM-1expression in FK(r =0.942, P =0.000).CONCLUSION: TREM-1 may contribute to amplify theinflammation in the cornea infected with A. fumigatus and play critical roles in the battle against A. fumigatus in the innate immune responses.

Boxb mediate BALB/c mice corneal inflammation through a TLR4/MyD88-dependent signaling pathway in Aspergillus fumigatus keratitis

AIM： To investigate whether high-mobility group box 1（HMGB1） Boxb exacerbates BALB/c mice corneal immune responses and inflammatory through the Toll-like receptor 4（TLR4）/myeloid differentiation primary response 88（My D88）-dependent signaling pathway in Aspergillus fumigatus（A. fumigatus） keratitis.METHODS： The mice corneas were pretreated with phosphate buffer saline（PBS）, Boxb before A. fumigatus infection. The abdominal cavity extracted macrophages were pretreated with PBS, Boxb, TLR4 inhibitor（CLI-095）, Dimethyl sulfoxide（DMSO） separately before A. fumigatus hyphae stimulation. HMGB1 was detected in normal and infected mice corneas and macrophages by real-time reverse transcriptase polymerase chain reaction（RT-PCR）, the TLR4, My D88, interleukin-1β（IL-1β）, tumor necrosis factor-α（TNF-α） were detected by Western blot and PCR.RESULTS： In BALB/c mice corneas, the expressions of TLR4, HMGB1, IL-1β, TNF-α were increased after A. fumigatus infection. While pretreatment with Boxb significantly increased the expressions of TLR4, HMGB1, My D88, IL-1β, TNF-α compared with PBS control after infection. In BALB/c mice abdominal cavity extracted macrophages, pretreatment with Boxb increased the expressions of TLR4, HMGB1, My D88, IL-1β, TNF-α, while pretreatment with CLI-095 and Boxb significantly decreased the expressions of TLR4, HMGB1, My D88, IL-1β, TNF-α. CONCLUSION： In A. fumigatus keratitis, Boxb play a proinflammatory role in corneal anti-fungi immune response through the HMGB1-TLR4-My D88 signal pathway.

Inhibitory Effect of Wood Vinegar Produced from Apricot Shell on Aspergillus fumigatus

[Objectives] This study was conducted to explore the antifungal activity of wood vinegar produced from apricot shell. [Methods]The minimal inhibitory concentration（ MIC） of wood vinegar against Aspergillus fumigatus was determined on agar plates. Then,the mycelial growth and the morphology of the apical vesicles of A. fumigatus cultured in the medium supplemented with 1/2 MIC wood vinegar were observed to evaluate the effect of wood vinegar on A. fumigatus.RNA-Seq was performed to explore the antifungal mechanism of wood vinegar. [Results] The wood vinegar produced from apricot shell could inhibit the growth of A. fumigatus,with a MIC of 20 μl/ml. In the medium supplemented with 1/2 MIC wood vinegar,the mycelial growth of A. fumigatus was suppressed,some apical vesicles were incompletely developed. The gene ontology（ GO） term enrichment analysis based on RNA-Seq showed that the differentially expressed genes（ DEGs）were enriched in metabolic process,cellular process and single-organism process in the category of biological processes,cell,cell part,membrane,organelle and membrane part in the category of cellular components,catalytic activity and binding in the category of molecular function. The significantly up-regulated DEGs were mostly involved in cell,cell part,organelle,organelle part and macro molecular complex in the category of cellular component,while the significantly down-regulated DEGs were mostly involved in biological process,cellular process and single-organism process in the category of biological process,catalytic activity,binding,transporter activity,and nucleic acid binding transcription factor activity in the category molecular function. Moreover,KEGG（ Kyoto Encyclopedia of Genes and Genomes） pathway analysis showed that the DEG unigenes involved in metabolism were the most,followed by those involved in genetic information processing.[Conclusions]The results indicated that the wood vinegar produced from apricot shell might inhibit the growth of A. fumigatus by interfering with the expression of multiple genes involved in multiple signaling pathways.

Thymol Protects against Aspergillus Fumigatus Keratitis by Inhibiting the LOX-1/IL-1βSignaling 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-1β,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-1β.Similarly,in vitro experiments indicated that the production of LOX-1 and IL-1βwas 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-1βsignaling pathway and reducing the recruitment of neutrophils and macrophages.

Dectin-1 agonist curdlan modulates innate immunity to Aspergillus fumigatus in human corneal epithelial cells

· AIM: To explore the immunomodulatory effects of curdlan on innate immune responses against Aspergillus fumigatus(A. fumigatus) in cultured human corneal epithelial cells(HCECs), and whether C-type lectin receptor Dectin-1 mediates the immunomodulatory effects of curdlan.·METHODS: The HCECs were stimulated by curdlan in different concentrations(50, 100, 200, 400 μg/m L) for various time. Then HCECs pretreated with or without laminarin(Dectin-1 blocker, 0.3 mg/m L) and curdlan were stimulated by A. fumigatus hyphae. The m RNA and protein production of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6) were determined by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The protein level of Dectin-1 was measured by Western blot.· RESULTS: Curdlan stimulated m RNA expression of TNF-α and IL-6 in a dose and time dependent manner in HCECs. Curdlan pretreatment before A. fumigatus hyphae stimulation significantly enhanced the expression of TNF-α and IL-6 at m RNA and protein levels compared with A. fumigatus hyphae stimulation group(P 【0.05).Both curdlan and A. fumigatus hyphae up-regulated Dectin-1 protein expression in HCECs, and Dectin-1expression was elevated to 1.5- to 2-fold by curdlan pretreatment followed hyphae stimulation. The Dectin-1blocker laminarin suppressed the m RNA expression and protein production of TNF-α and IL-6 induced by curdlan and hyphae(P 【0.05).· CONCLUSION: These findings demonstrated that curdlan pretreatment enhanced the inflammatory response induced by A. fumigatus hyphae in HCECs.Dectin-1 is essential for the immunomodulatory effectsof curdlan. Curdlan may have high clinical application values in fungal keratitis treatment.

Indoleamine 2,3-dioxygenase adjusts neutrophils recruitment and chemotaxis in Aspergillus fumigatus keratitis

AIM: To explore the effect of indoleamine 2,3-dioxygenase(IDO) on recruitment and chemotaxis function of neutrophils in Aspergillus fumigatus(A.fumigatus) keratitis.METHODS: C57BL/6 mice models of A.fumigatus keratitis were established by inoculating hyphae of A.fumigatus evenly on the corneas.The clinical scores and inflammatory cytokines expression were measured respectively on the 1^(st), 3^(th), 5^(th) day after infection.The 1-MT(1 mg/m L) was administered by gavage to exert an inhibitory effect on IDO during infection.The mice were divided into control group, 1-MT group, A.fumigatus(A.F.) group, and 1-MT+A.F.groups.The corneas were monitored by slit lamp microscopy, and recorded disease scores in 3 d after infection.Myeloperoxidase(MPO) assay was done to evaluate the neutrophils infiltration.Immunofluorescence staining was used to detect the recruitment of neutrophils in murine corneas.The m RNA of inflammatory cytokines was measured with reverse transcription-polymerase chain reaction(RT-PCR).RESULTS: The corneal inflammation and the clinical score reached the peak on the 3;day after the corneal infection.The m RNA of inflammatory cytokines of the A.F.group reached the highest on the 3;day after the infection accordingly.Meanwhile, the results of slit light photography indicated that inhibitors of IDO made inflammation more serious contrasted with the A.F.group on the 3;day.Besides, imunofluorescence staining and MPO indicated that 1-MT enhanced the recruitment, infiltration and chemotaxis of neutrophils obviously in contrast to the A.F.group.RT-PCR indicated that 1-MT increased the expression of CXCL-1, ICAM-1, IL-1β, and IL-8 significantly.CONCLUSION: IDO participates in the pathogenesis of A.fumigatus keratitis and plays an important role in inducing immune protection by inhibiting neutrophils-related inflammatory reaction and suppressing recruitment and chemotaxis of the neutrophils.

红树林内生真菌Aspergillus fumigatus SAS10二聚萘并吡喃酮类代谢产物研究

通过大米培养基静置发酵无瓣海桑内生真菌Aspergillus fumigatus SAS10,采用硅胶柱色谱、Sephadex LH-20凝胶柱色谱以及半制备高效液相色谱等分离手段对其发酵产物进行分离纯化,获得8个纯化合物,其结构经核磁共振波谱、质谱等现代分析方法,分别鉴定为asperpyrone A(1)、aurasperone B(2)、asperpyrone C(3)、asperpyrone D(4)、dianhydro-aurasperone C(5)、aurasperone A(6)、fonsecinone A(7)和rubrofusarin B(8)。结构鉴定结果显示,化合物1~8均为首次从烟曲霉中分离得到,菌株Aspergillus fumigatus SAS10具有产生大量二聚萘并吡喃酮类代谢产物的潜力。抑菌活性表明,化合物1~8对金黄色葡萄球菌、大肠杆菌和耐甲氧西林金黄色葡萄球菌具有不同程度的抑制活性。