Butyl alcohol extract of Baitouweng Decoction alleviates vulvovaginal candidiasis in mice by downregulating NLRP3 inflammasome and related signal pathways / 中国中药杂志

This study aims to explore the effect of butyl alcohol extract of Baitouweng Decoction(BAEB) on vulvovaginal candidiasis(VVC) in mice and to clarify the mechanism from Toll-like receptors(TLRs)/MyD88 and Dectin-1/Syk signal pathways and NLRP3 inflammasome. To be specific, female KM mice were randomized into control group(i.g., normal saline), model group, fluco-nazole group(i.g., 20 mg·kg~(-1)), and low-dose, medium-dose, and high-dose BAEB groups(i.g., 20, 40, and 80 mg·kg~(-1), respectively). VVC was induced in mice except the control group. After the modeling, administration began and lasted 7 days. The ge-neral conditions and body weight of mice were recorded every day. On the 1 st, 3 rd, 7 th, and 14 th after vaginal infection by Candida albicans, the fungal load in the vaginal lavage fluid of the mice was measured with the plate method, and the morphology of C. albicans in vaginal lavage fluid was observed based on Gram staining. After the mice were killed, vaginal tissues were subjected to hematoxylin-eosin(HE) staining and periodic acid-Schiff(PAS) staining for vaginal histopathological analysis. The content of cytokines in vaginal lavage fluid, such as interleukin(IL)-1β, IL-18, tumor necrosis factor-α(TNF-α), IL-6, and S100 a8, was determined by enzyme-linked immunosorbent assay(ELISA), and content of reactive oxygen species(ROS) in vaginal tissues by tissue ROS detection kit. The protein expression of NLRP3, ASC, caspase-1, Dectin-1, Syk, MyD88, TLR2, TLR4, and nuclear factor-κB(NF-κB) in vaginal tissues was detected by Western blot, and the levels and distribution of NLRP3, Dectin-1, Syk, MyD88, TLR2, and TLR4 in vaginal tissues were determined with the immunohistochemical method. The results show that BAEB can improve the general conditions of VVC mice, reduce the fungal load and C. albicans hyphae in vaginal secretion, decrease ROS content in vaginal tissues and content of cytokines in vaginal lavage fluid, and down-regulate the expression of NLRP3, ASC, caspase-1, Dectin-1, Syk, MyD88, TLR2, TLR4, and NF-κB in vaginal tissues. The above results indicate that BAEB exerts therapeutic effect on VVC mice by down-regulating the key proteins in the TLRs/MyD88 and Dectin-1/Syk signal pathways and NLRP3 inflammasome.

Therapeutic effect of cinnamaldehyde on ulcerative colitis in mice induced by dextran sulfate sodium with Candida albicans colonization and its effect on dectin-1/TLRs/NF-κB signaling pathway / 中国中药杂志

To observe the efficacy of cinnamaldehyde on dextran sulfate sodium(DSS)-induced ulcerative colitis(UC) with Can-dida albicans(Ca) colonization and its effect on dectin-1/TLRs/NF-κB signaling pathway in mice. C57 BL/6 mice were randomly divided into normal group, DSS group, DSS+Ca group, cinnamaldehyde group and mesalazine group. Mice in DSS+Ca group were given Ca(1×10~8 CFU per mouse) through intragastrical administration for 4 consecutive days and then distilled water with 3.0% DSS for 7 consecutive days. In cinnamaldehyde group and mesalazine group, in addition to the induction method of the DSS+Ca group, mice were given 75 mg·kg~(-1) cinnamaldehyde and 200 mg·kg~(-1) mesalazine accompanied with 3.0% DSS for 7 consecutive days, respectively. Mice in normal group and DSS group were correspondingly administered with distilled water. The general conditions of the mice were observed daily, the diseased activity index(DAI) score was calculated, and fungal loads of feces were detected by plate method. The mice were sacrificed on day 12, colon length was measured, colon mucosa damage index(CMDI) score was calculated, and histopathological analysis was carried out by HE staining. Anti-saccharomces cerevisiae antibody(ASCA) and β-1,3-glucan in serum, and TNF-α, IL-1β, IL-6, IL-8, IL-10 in serum and colon tissue were detected by ELISA. The contents of β-1,3-glucan and macrophage infiltration in colon tissues were examined by immunofluorescence staining. The protein expressions of dectin-1, TLR2, TLR4 and NF-κB were detected by Western blot and immunohistochemistry staining. The results showed that cinnamaldehyde could significantly improve the general conditions of UC mice with Ca colonization, decrease DAI and histopathological scores, reduce intestinal mucosal congestion, erosion and colon shortening, decrease Ca load in mouse feces and tissues, down-regulate the contents of ASCA and β-1,3-glucan in serum, reduce the contents of TNF-α, IL-1β, IL-6, IL-8 and increase IL-10 in serum and colon tissues, inhibit macrophages infiltration and down-regulate the protein expression of dectin-1, TLR2, TLR4 and NF-κB in colon tissue. These results suggested that cinnamaldehyde had a therapeutic effect on UC mice with Ca colonization, which might be related to the inhibition of Ca proliferation, the regulation of dectin-1/TLRs/NF-κB signaling pathways and the coordination of the balance between pro-inflammatory and anti-inflammatory factors.

Association between polymorphisms in the genes encoding toll-like receptors and dectin-1 and susceptibility to invasive aspergillosis: a systematic review

Abstract Invasive aspergillosis is a common fungal infection in immunocompromised individuals. Some studies have shown that toll-like receptor and dectin-1 genetic polymorphisms may alter signaling pathways, thus increasing an individual's susceptibility to invasive aspergillosis. We investigated the pertinent literature to determine whether polymorphisms in the genes encoding toll-like receptors and dectin-1 increase the susceptibility to invasive aspergillosis. This study systematically reviewed the literature using the databases PubMed/PMC, Scopus, and Web of Science using the keywords invasive aspergillosis, polymorphism, Toll-like, and Dectin-1. From the initial search, 415 studies were found and according to our inclusion and exclusion criteria, eight studies were selected. Several studies described single-nucleotide polymorphisms (SNPs) that are associated with a greater susceptibility to invasive aspergillosis. These SNPs were found in the genes that encode toll-like receptors 1, 3, 4, and 5 and the gene that encodes dectin-1; upon activation, both cellular receptors initiate a signaling cascade that can result in the production of cytokines and chemokines. Thus, our literature review uncovered a significant association between polymorphisms in the genes that encode toll-like receptors and dectin-1 and invasive aspergillosis. More studies should be performed to better understand the relationship between toll-like receptor and dectin-1 genetic polymorphisms and invasive aspergillosis susceptibility.

Heat-Killed Saccharomyces cerevisiae, A Dectin-1 Agonist, Selectively Induces IgG4 Production by Human B Cells

Dectin-1 is a major receptor that recognizes fungal cell wall β-glucan. We previously reported that heat-killed Saccharomyces cerevisiae (HKSC), a Dectin-1 agonist, selectively induces IgG1 class switching in mouse B cells. Dectin-1 is also expressed on human B cells; however, Dectin-1 function in human B cells remains unknown. This study aimed to investigate the direct effect of in vitro stimulation using HKSC on Ig class switching in human B cells. HKSC selectively induced the expression of germline γ4 transcripts (GLTγ4) by human B cell line 2E2, and HKSC significantly augmented GLTγ4 promoter activity. Moreover, HKSC selectively enhanced GLTγ4 expression and IgG4 production by anti-CD40-activated human tonsillar resting B cells. Thus, these results suggest that Dectin-1 maybe involved in selective IgG4 class switching by human B cells.

Roles of Dectin-1 in phagocytosis of Candida albicans by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1 / 中华皮肤科杂志

Objective To investigate the roles of Dectin-1 in phagocytosis of Candida albicans (C.albicans) by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1.Methods THP-1 macrophage-like cells served as the target cells,and were transfected with small interfering RNA (siRNA) targeting Dectin-1 to down-regulate the expression of Dectin-1 receptor (siRNA-Dectin-1 group).THP-1 macrophage-like cells transfected with nonsense siRNA (siRNA-NC) served as a negative control group.After transfection,the THP-1 macrophage-like cells in the above 2 groups were cocultured with heat-killed C.albicans separately.And then,fluorescence microscopy was performed to count THP-1 macrophage-like cells phagocytosing C.albicans,and flow cytometry was used to determine the mean fluorescence intensity (MFI) of dihydrorhodamine (DHR)-123 fluorescent cells.Statistical analysis was done by one-way analysis of variance (ANOVA) and t test with the SPSS19.0 software.Results After transfection with siRNA-Dectin-1,the mRNA and protein expression of Dectin-1 significantly decreased in THP-1 macrophage-like cells (t =26.163,P ＜ 0.001).After 1-,2-,4-hour co-culture of THP-1 macrophagelike cells with C.albicans,fluorescence microscopy showed that the phagocytosis rates of C.albicans by THP -1 macrophage-like cells were significantly lower in the siRNA-Dectin-1 group than in the negative control group (17.5％ vs.22.1％,18.6％ vs.24.3％,39.2％ vs.59.1％,respectively,all P ＜ 0.05),so were the percentage of THP-1 macrophage-like cells phagocytosing more than 3 C.albicans cells (2.2％ vs.4.7％,2.5％ vs.5.4％,5.1％ vs.8.3％,respectively,all P ＜ 0.05).After 30-minute,1-,2-and 4-hour co-culture of THP-1 macrophage-like cells with DHR-123-labelled C.albicans,flow cytometry showed that the MFI of C.albicans-phagocytosing cells was significantly lower in the siRNA-Dectin-1 group than in the negative control group (36.8 vs.45.7,54.3 vs.62.4,72.1 vs.84.9,93.6 vs.116.7,respectively,all P ＜ 0.05).Conclusion Dectin-1 receptor plays an important role in the phagocytosis of C.albicans by macrophages.

β-(1,3)-Glucan derived from Candida albicans induces inflammatory cytokines from macrophages and lamina propria mononuclear cells derived from patients with Crohn's disease

BACKGROUND/AIMS: Recent research has highlighted the importance of interactions between commensal fungi and intestinal inflammation. However, there are few studies investigating whether commensal fungi contribute to inflammation in patients with Crohn's disease (CD). The aim of this study is to investigate reveal interactions between commensal fungi and host immune cells in CD. METHODS: CD14-positive monocytes were isolated from peripheral blood mononuclear cells from healthy human volunteers and then differentiated in the presence of macrophage colony-stimulating factor (M-CSF) (referred to as M-macrophages, M-Mϕs) or M-CSF and interferon-γ (IFN-γ) (referred to as M-gamma macrophages, Mγ-Mϕs). Cytokine production by these in vitro differentiated macrophages in response to β-(1,3)-glucan was analyzed by flow cytometry. Expression of Dectin-1 was examined using flow cytometry, western blotting, and quantitative reverse transcription-polymerase chain reaction. Cytokine production by in vitro differentiated macrophages in response to β-(1,3)-glucan was measured in the presence of an anti-Dectin-1 receptor antagonist, anti-Syr, or an anti-Fas-1 antibody. Cytokine production by lamina propria mononuclear cells (LPMCs) derived from CD patients in response to β-(1,3)-glucan was also analyzed. RESULTS: Mγ-Mϕs produced a large amount of tumor necrosis factor-α (TNF-α) and interleukin-6 in response to β-(1,3)-glucan. Dectin-1 expression was significantly higher in Mγ-Mϕs than in M-Mϕs. The increase in TNF-α production by Mγ-Mϕs stimulated with glucan was reversed by blocking Dectin-1, Syr or Fas-1. LPMCs derived from CD patients stimulated with β-(1,3)-glucan produced significantly higher amount of TNF-α than LPMCs derived from UC patients. CONCLUSIONS: These results suggest that commensal fungal microbiota may contribute to the pathogenesis of CD by inducing macrophages-derived pro-inflammatory cytokines.

Role of Dectin-1 in the production of IL-10 and TNF-α by rat tracheal epithelial cells stimulated with heat-treated Candida glabrata / 中国感染控制杂志

Objective To investigate the effect of Dectin-1 on the release of inflammatory factors interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) in rat tracheal epithelial cells (RTECs) stimulated with heat-treated Candida glabrata (C.glabrata).Methods RTECs cultivated in vitro were randomly divided into three groups,including control group(RTECs + sterile normal saline),fungal stimulation group(RTECs + heat-treated C.glabrata),and inhibitor intervention group(RTECs + laminarin + heat-treated C.glabrata),cells were harvested after incubation for 0,2,4,6 hours respectively,cell survival rate was determined by MTT method,expression of Dectin-1 was analyzed by Western Blot,expression of IL-10 and TNF-α were detected by enzyme-linked immunosorbent assay (ELISA).Results Heat-treated C.glabrata destroyed cell structure and reduced cell survival rate.At the beginning of the culture (0 h),cell survival rate,expressions of Dectin-1,IL-10 and TNF-α among three groups were all not significantly different(all P＞0.05).After incubation for 2,4,6 hours,expressions of Dectin-1,IL-10 and TNF-α in fungal stimulation group and inhibitor intervention group were both significantly higher than control group;expressions of Dectin-1,IL-10,and TNF-α in inhibitor intervention group was lower than fungal stimulation group(all P＜0.05).The expressions of IL-10 in inhibitor intervention group at 0 h and 2 h was not significantly different,expressions of Dectin-1,IL-10,and TNF-α in fungal stimulation group and inhibitor intervention group at different incubation periods were significantly different(all P＜0.05).Conclusion Dectin-1 is an important receptor for RTECs to recognize the heat-treated C.glabrata,it induces the release of IL-10 and TNF-α,and mediate the occurrence of inflammation.

Effects of immunosuppressants on Dectin-1 and TLR2 expression in macrophages / 中国免疫学杂志

Objective:To study the effects of dexamethasone,cyclophosphamide,cyclosporin A and mycophenolate mofetil on the expression of Dectin-1 and TLR2 receptor in RAW264.7 cell line.Methods:In vitro culturing RAW264.7 macrophages ,the cells were given different doses of dexamethasone and cyclophosphamide ,cyclosporin A and mycophenolate mofetilfor 24 h.The influence of different immunosuppresants on cell proliferation was detected by CCK-8 assay.Dectin-1,TLR2 mRNA and protein levels of change were detected with RT-PCR technique and flow cytometry .Results:CCK-8 cell proliferation toxicity experiment results showed that with the increase of the dose of dexamethasone ,cyclophosphamide ,Cyclosporin A and mycophenolate mofetil ,the different degree of inhibi-tion of RAW264.7 cell proliferation in mice can be saw after the intervention of 24 h (P0.05 ) .Mycophenolate mofetil and cyclosporin A could both inhibit the expression of Dectin-1 and TLR2 (P<0.05).Conclusion:There are significant differences among the regulatory functions of various immunosupres -sants on the expression of pattern recognition receptors .Different immunosuppressive agents exert their inhibitory effects on immune rec-ognition process of fungal pathogens not only by selectively regulating their target PRRs expression ,but also by inhibiting the growth and proliferation of macrophages .

Effects of heat-treated candida glabrata on the expression of Dectin-1,the production of IL-6 and TNF-α in rat tracheal epithelial cells / 重庆医学

Objective To explore the effects of co‐cultured heat‐treated candida glabrata with rat tracheal epithelial (RTE) cells on the expression of Dectin‐1 and the production of IL‐6 and TNF‐α.Methods RTE cells in vitro were co‐cultured with heat‐treated candida glabrata bacteria liquid for 2 ,4 ,6 h ,while without co‐cultured RTE cells were used as control group .We observed the morphological changes of RTE cells ,detected the protein expressions of Dectin‐1 by Western blot ,used real‐time PCR to detecte the mRNA expressions of IL‐6 and TNF‐αand measured protein expression of IL‐6 and TNF‐αby ELISA .Results With the pass‐ing of time ,the RTE cells were damaged extensively and the expression of Dectin‐1 ,IL‐6 and TNF‐αbecame more and more signifi‐cant .Obviously ,there had significant difference in the expression of Dectin‐1 ,IL‐6 and TNF‐α between the co‐cultured 2 h group and the control group ,the co‐cultured 4 h group and the co‐cultured 2 h group ,the co‐cultured 6 h group and the co‐cultured 4 h group (P<0 .05) .Conclusion RTE cells have natural immune function .The Dectin‐1 involves in the recognition of heat‐treated Candida glabrata ,activating secretion of IL‐6 and TNF‐αand mediating inflammatory reaction .IL‐6 plays a negative regulation role .

Regulation of Toll-like receptors in fungal keratitis / 中华实验眼科杂志

Fungal keratitis (FK) ,a potential blinding disease,has been difficult to treat due to the limited number of approved antifungal drugs and the taxing dosing regimen.Toll-like receptors (TLRs) function as the pattern recognition receptors (PRRs) in mammals and play an essential role in the recognition of fungal components.There is a great amount of evidence that TLRs initiate the innate immunity in the FK.Furthermore, TLRs also play roles in shaping fungal-specific humoral and cellular adaptive immune responses.This review described the recent advances in interaction between TLRs and non-TLRs signal transduction,negative regulator and function of TLRs in corneal fungal infection.

Construction and identification of a recombinant adenoviral vector expressing murine dendritic cell-associ-atedC-type lectin-1 / 医学研究生学报

Objective Dendritic cell-associatedC-type lectin-1 ( Dectin-1) is one of the most important receptors in antifungal innate immune response.This study was to construct a recombinant adenovirus vector expressing themurine Dectin-1gene and acquire a high-concentration adenovirus by amplification and purification. Methods The PCR amplification product CLEC7A-pIRES2-EGFP was cloned into the intermediate vector pDONR221, and then recom-bined with the backbone vector pAD/CMV/V5-DEST to produce a re-combinant plasmid pAD-CLEC7A-pIRE2S -EGFP.The recombinant plasmid was linearized with Pac I and transfected into human embryon-ic kidney ( HEK293) cells to produce recombinant adenovirus pAD-CLEC7Ap-IRES 2-EGFP. The adenovirus was propagated in the HEK293 cells and purified by filtering through the cellulose acetate membrane and concentrating column.Fluorescence microscopy and re-al-time PCR were used to determine the expression of the Dectin-1 gene. Results PCR identification, enzyme digestion, and sequen-cing results manifested theDectin-1 gene in the vector, with the final adenovirus titer of 5×1011 IU/mL.Fluorescence microscopy revealed green fluorescence and real-time PCR assay confirmed that the expression of Dectin-1 was improved by 8677.25 times. Conclusion A relatively high-titer adenovirus expressing Dectin-1 was acquired,which may help to further study the high expression of Dectin-1 in anti-fungal innate immunity in vitro and in vivo.

Expressions of TLR4 and Dectin-1 in cornea tissue of rats with Aspergillus fungal keratitis and their effects in pathogenesis of keratitis / 吉林大学学报(医学版)

Objective To explore the expressions of Dectin-1 and Toll-like receptor 4 (TLR4)and the secretory charateristics of inflammatory factors of the Aspergillus fumigatus infected cornea of the rats,and to clarify theirs role in the pathogenesis of rat keratitis.Methods 40 SD rats were separated into control group (n = 10)and aspergillus keratitis group (n=30).The rats in aspergillus keratitis group were infected by Aspergillus fumigatus in two eyes for 12,24 and 48 h,and the cornea tissue was took and ELISA kit was used to detect the level of inflammatory cytokines IL-1,TNF-a,IL- 6,IL-10,and NF-κB;immunohistochemistry,Western blotting and RT-PCR methods were used to determine the Dectin-1 and TLR4 protein and mRNA expressions.Results The rat models were successfully established.The eyes of the rats in control group were limpid tested by corneal slit lamp, however,there existed a clear boundary film on the surface of eyes of the rats in aspergillus keratitis group infected for 12 h,white infiltrates and bloodshot in the eyes in 24 h group and the white infiltrates were further strengthened and almost covered the entire eyes in 48 h group. The order of inflammatory cytokines IL-1,TNF-α,IL-6,IL-10, NF-κB was:48 h group> 24 h group> 12 h group> control group (P < 0.05),so did the expression levels of Dectin-1 and TLR4 protein and mRNA in various groups tested by Western blotting and RT-PCR methods.The expression levels of Dectin-1 and TLR4 were increased with the prolongation of time investigated by immunohistochemical staining.Conclusion Dectin-1 and TLR4 are secreted in excess after the eyes are infected with fumigatus,which promotes the expressions of related inflammatory cytokines and results in the occurrence of keratitis.

The change of serum IL-17 and Dectin-1 in non-neutropenic patients with invasive pulmonary aspergillosis / 中国感染与化疗杂志

Objective To explore the changing levels of serumIL-17 and Dectin-1 and their implication in non-neutropenic pa-tients with invasive pulmonary aspergillosis (IPA).Methods The clinical data were reviewed for 23 non-neutropenic patients with clinical diagnosis of IPA (IPA group),31 patients with clinical diagnosis of pneumonia (pneumonia group)and 51 healthy subjects(control group).The peripheral serum was collected to analyze IL-17 level by ELISA.Serum Dectin-1 level was also determined at the same time.Serum G test,GM test,WBC and CRP level were also assayed for the patients in IPA group.Pa-tient outcome was followed up and analyzed in terms of serum IL-17.Results The serum IL-17 level of the patients in IPA group was significantly higher than that in the control group (P 0.05).The serum Dectin-1 level in IPA group was significantly higher than that in the control group (P 0.05 ). Conclusions Dectin-1 may be produced and Th17 cell immunity activated in response to Aspergillus fumigatus in-fection in non-neutropenic patients.And,serum Dectin-1 level is correlated with Th17 response.

Co-stimulation of TLR4 and Dectin-1 Induces the Production of Inflammatory Cytokines but not TGF-beta for Th17 Cell Differentiation

Collaboration of TLR and non-TLR pathways in innate immune cells, which acts in concert for the induction of inflammatory cytokines, can mount a specific adaptive immune response tailored to a pathogen. Here, we show that murine DC produced increased IL-23 and IL-6 when they were treated with LPS together with curdlan that activates TLR4 and dectin-1, respectively. We also found that the induction of the inflammatory cytokine production by LPS and curdlan requires activation of IKK. However, the same treatment did not induce DC to produce a sufficient amount of TGF-beta. As a result, the conditioned media from DC treated with LPS and curdlan was not able to direct CD4+ T cells to Th17 cells. Addition of TGF-beta but not IL-6 or IL-1beta was able to promote IL-17 production from CD4+ T cells. Our results showed that although signaling mediated by LPS together with curdlan is a potent stimulator of DC to secrete many pro-inflammatory cytokines, TGF-beta production is a limiting factor for promoting Th17 immunity.

Serum Dectin-1 in non-agranulocytic patients with pulmonary aspergillosis / 中国感染与化疗杂志

Objective To explore the clinical implication of serum Dectin-1 level in the non-agranulocytic patients with pulmonary aspergillosis. Methods Serum specimen were collected from the non-agranulocytic patients with pulmonary aspergillosis to determine the serum level of Dectin-1 with ELISA.The relationship between serum Dectin-1 level,the results of G test and galactomannan (GM)test of Aspergillus,and white blood cell count was analyzed.Results The serum Dectin-1 level was (427.2 ± 42.6)pg/mL in the patients with Aspergillus infection,and (280.8 ± 39.4)pg/mL in the control patients (P<0.05 ).Dectin-1 level was not correlated to white blood cell count,or the result of G test,or GM test. Conclusions Serum Dectin-1 level increases significantly in the non-agranulocytic patients with pulmonary aspergillosis, suggesting that Dectin-1 is an important anti-Aspergillus immune molecule.

Dectin-1 Stimulation Selectively Reinforces LPS-driven IgG1 Production by Mouse B Cells

Dectin-1, which specifically recognizes beta-glucan of fungal cell walls, is a non-Toll-like receptor (TLR) pattern recognition receptor and a representative of C-type lectin receptors (CLRs). The importance of Dectin-1 in innate immune cells, such as dendritic cells and macrophages, has previously been well studied. However, the function of Dectin-1 in B cells is very poorly understood. To determine the role of Dectin-1 in B cell activation, we first investigated whether mouse B cells express Dectin-1 and then assessed the effect of Dectin-1 stimulation on B cell proliferation and antibody production. Mouse B cells express mRNAs encoding CLRs, including Dectin-1, and surface Dectin-1 was expressed in B cells of C57BL/6 rather than BALB/c strain. Dectin-1 agonists, heat-killed Candida albicans (HKCA) and heat-killed Saccharomyces cerevisiae (HKSC), alone induced B cell proliferation but not antibody production. Interestingly, HKSC, HKCA, and depleted zymosan (a selective Dectin-1 agonist) selectively enhanced LPS-driven IgG1 production. Taken together, these results suggest that, during fungal infection, beta-glucan-stimulated Dectin-1 may cooperate with TLR4 to specifically enhance IgG1 production by mouse B cells.

G-test positive serum inhibits ROS-dependent killing of Candida albicans by interfering with internal- ized expression of neutrophil Dectin-1 / 中华微生物学和免疫学杂志

Objective To investigate the mechanism of soluble β-1, 3-D-glucan in G-test positive serum in inhibiting ROS-dependent killing of Candida albicans ( C.albicans ) mediated by neutrophil Dectin-1.Methods The expression and distribution of internalized Dectin-1 and triggered ROS in human neutrophils were detected by using confocal/two-photon laser scanning microscopy upon stimulation with C.albicans (MOI=10) which was pretreated with β-1, 3-D-glucanase (10 U/ml) or not.Abrogation test was used to analyze whether intracellular Dectin-1 was involved in C.albicans-triggered ROS production in human neutrophils.Furthermore, flow cytometry analysis was performed to detect the expression of intracel-lular Dectin-1 and ROS in neutrophils which were pretreated respectively with G-test positive serum at differ-ent dilutions for 60 min and then stimulated with C.albicans for another 60 min at 37℃.Results After stimulated with C.albicans (MOI=10) for 60 min, the expression of Dectin-1 in neutrophils was recruited to the spores of opsonophagocytized C.albicans, and partly co-localized with the triggered ROS production . However, the expression of intracellular Dectin-1 was not observed in neutrophils when stimulated with β-1, 3-D-glucanase pretreated C.albicans for 60 min at 37℃.Abrogation test further showed that C.albicans-trig-gered ROS production in neutrophils was partly and irreversibly inhibited by adding Dectin -1 blocking mAb of 5 μg/ml.In addition , both the triggered expression of intracellular Dectin-1 and ROS production in neu-trophils stimulated with C.albicans ( MOI=10 ) in the presence of G-test positive serum were significantly lower than those of neutrophils stimulated only with C.albicans (LSD-t test, P<0.01).Linear regression a-nalysis suggested that the triggered intracellular Dectin-1 and ROS production in neutrophils upon stimulation with C.albicans were both inhibited by soluble β-1, 3-D-glucan in a dose-dependent manner (Dectin-1,R2=0.702,P<0.01;ROS,R2=0.588,P<0.01 ).Conclusion Taken together, these results demonstrated that the soluble β-1, 3-D-glucan in G-test positive serum may play a role in inhibiting the ROS-dependent killing of C.albicans by interfering with internalized expression of neutrophil Dectin-1.

Molecular half-full mechanisms by phagocityc cells on invasive Aspergilosis / Mecanismos moleculares mediados por celulas fagociticas en la aspergilosis invasiva

Fungus from the Aspergillus genus mainly affects lung tissue, occurring when the integrity of the host immunesystem is compromised. The human body uses immunocompetence conditions from mechanical and enzymatic defenses and the action of the innate immune system cells and also uses adaptive responses to control infection. Neutrophils, macrophages, and dendritic cells are critical as antifungal effector cells possess surface receptors that recognize fungal structures and trigger specific responses. TLRs and Dectin-1 the most studied for this interaction. TLRs are responsible for the production and release of cytokines and Dectin-1 is essential in the phagocytosis of theparticle recognition and production of ROS. The best-studied cytokines and its crucial role in the response toAspergillus spp. are TNF-á, IFN-ã, and IL-12. In this work, we reviewed the main mechanisms related to molecularreceptors on phagocytic cells involved in the recognition of Aspergillus spp. Understanding the immune response insituations of immunocompetence and its comparison in immunodeficient organisms could provide alternatives tocontrol invasive aspergillosis.

Intracellular expression of Dectin-1 mediates the killing of Candida albicans by human neutrophils in a manner dependent on ROS production / 中华微生物学和免疫学杂志

Objective To investigate the mechanism hy which Dectin-1 mediates the killing of Candida albicans(C.albicans) by human neutrophils in a manner dependent on the production of reactive oxygen species (ROS).Methods After stimulation with FITC-C.albicans at a multiplicity of infection (MOI) of 10 for 30 or 60 min,PE-anti-human Dectin-1 monoclonal antibody (2.5 μg/106 cells) was used to detect the expression of Dectin-1 in human neutrophils by flow cytometry.For Dectin-1 inhibition test and ROS assay,human neutrophils (2×106/ml) were respectively pre-incubated with different concentrations of blocking antibody (0.5,1,2.5 and 5 μg/ml) for 60 min at 4℃,and then with 25 μmol/L 2′,7′-Dichlorofluorescein diacetate for another 20 min at room temperature.Afterwards,under stimulation with live C.albicans at a MOI of 10,the rate of intracellular ROS production over time in blocking and control groups was measured continuously at 10 min intervals for up to 120 min.In addition,localization of Dectin-1 and ROS in human neutrophils was observed by confocal/two-photon laser scanning microscopy after stimulation with live C.albicans.For the detection of candicidal activity,after pre-treatment with different concentrations of Dectin-1 blocking antibody as mentioned above,neutrophils were stimulated with live C.albicans (MOI=1) for 60 min,serial dilutions of cell lysate were plated onto yeast agar,and CFU were enumerated after incubation at 37℃ for 48 h.The candicidal activity was represented as [1-(CFUblocking group/CFUbuffer)] × 100％.Results Under stimulation with FITC-C.albicans at a MOI of 10 for 30 and 60 min,positive percentage of intracellular Dectin-1-expressing neutrophils increased significantly when compared with initial level (0 min,8.32％ ; 30 min,16.82％ ; 60 min,23.88％) (versus 0 min,P＜0.01).However,positive percentage of cell-surface Dectin-1-expressing neutrophils remained almost unchanged after stimulation for 30 and 60 min (versus 0 min,P＞0.05).Upon blockage of Dectin-1,the stimulated ROS generation (R2=0.306,P＜0.01) and candicidal activity (R2=0.251,P＜0.01) of neutrophils were partly and irreversibly inhibited in a dose-dependent manner when compared with control group.In addition,the intracellular Dectin-1 is recruited and co-distributed with ROS on the surface of phagocytized C.albicans as observed by confocal microscopy.Conclusion Taken together,these results demonstrated that an internalized expression pattern of human Dectin-1 might contribute to the ROS-dependent killing of serum-opsonzied C.albicans which was phagocytized by human neutrophils.

Role of β-glucan receptor dectin-1 in anti-fungal infection of cornea / 中华实验眼科杂志

β-Glucan is a kind of highly conservative composition on fungal cell wall.As a recognition receptor of β-glucan,dendriticcell-associatedC-typelectin-1 (dectin-1)widely distributeinsingle-nuclea macrophages system,dendritic cells ( DC )and neutrophil cells.Through the identification of fungal cell wall composition and cells signal transduction,dectin-1 induces the production of cytokine and chemokine and then starts a nonspecific and specific immunity response.Dectin-1 plays a key role in antifungal immune response.Here,the molecular structure,tissue cells,signal transmission and the immune inflammation role of dectin-1 in corneal infection were reviewed.