Mannan-binding lectin inhibits oxidative stress-induced senescence via the NAD+/Sirt1 pathway.

Prolonged or excessive oxidative stress can lead to premature cellular and body aging. Mannan-binding lectin (MBL) is synthesized by the liver and plays an important role in innate immunity, anti-inflammation, and anti-oxidation, and has a positive impact on health and longevity. To date, few studies investigated the role of MBL in attenuating oxidative stress-induced senescence. In this study, we evaluated the role of MBL in oxidative stress-induced premature aging and explored its underlying mechanism in C57BL/6 mice and mouse embryonic fibroblasts (NIH/3T3). First, we established an oxidative premature senescence model induced by D-galactose in C57BL/6 mice. We found that MBL-deficient mice had a marked aging-like appearance, reduced learning and spatial exploration abilities, severe liver pathological damage, and significantly upregulated expression of Senescence-associated proteins (p53 and p21), inflammatory kinesins (IL-1ß and IL-6), and the senescence ß-galactosidase (SA-ß-Gal) positive rate as compared with WT mice. In the H2O2-induced oxidative senescence model of NIH/3T3 cells, consistent results were obtained after MBL intervention. In addition, MBL effectively inhibited G1 phase arrest, ROS levels, DNA damage, and mitochondrial dysfunction in premature senescent cells. Mechanistically, we found that oxidative stress inhibited the nicotinamide adenine dinucleotide (NAD+)/ silent information regulator 1 (Sirt1) signaling pathway, while MBL activated the NAD+/Sirt1 signaling pathway inhibited by oxidative stress. In addition, MBL could activate the NAD+/Sirt1 pathway by upregulating NAMPT, which in turn inhibited p38 phosphorylation by activating the NAD+/Sirt1 pathway. In conclusion, MBL inhibits oxidative aging, which may facilitate the development of therapeutics to delay oxidative aging.

ZnS/C Dual-Quantum-Dots Heterostructural Nanofibers for High-Performance Photocatalytic H2O2 Production.

Constructing heterostructures of dual quantum-dots (QDs) is a promising way to achieve high performance in photocatalysis, but it still faces substantial synthetic challenges. Herein, we developed an in situ transformation strategy to coassemble ZnS QDs and C QDs into dual-quantum-dot heterostructural nanofibers (ZnS/C-DQDH). Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy results revealed the formation of strong Zn-O-C bonds at the interface between ZnS QDs and C QDs, improving the separation efficiency of photogenerated charge carriers. The ZnS/C-DQDH demonstrated remarkable photocatalytic activity in H2O2 production, with generation rates of 2896.4 µmol gcat-1 h-1 without sacrificial agents and 9879.3 µmol gcat-1 h-1 with ethanol as the sacrificial agent, significantly higher than the QD counterparts and surpassed state-of-the-art photocatalysts. Moreover, due to the nanofibrous feature, ZnS/C-DQDH demonstrated excellent stability and facile recyclability. This work provides a facile and large scalable method to gain dual-quantum-dot heterostructures and a promising alternative for photocatalytic H2O2 production.

High expression of B4GALT1 is associated with poor prognosis in acute myeloid leukemia.

Acute myeloid leukemia is the most prevalent type of leukemia in adults and is prone to relapse and chemoresistance, with a low long-term survival rate. Therefore, the identification of quality biomarkers constitutes an urgent unmet need. High expression of beta-1,4-galactosyltransferase 1 (B4GALT1) has been observed in several cancer types; however, its function in acute myeloid leukemia has rarely been studied. Therefore, our study obtained gene expression data from The Cancer Genome Atlas (TCGA) database to analyze the relationship between B4GALT1 and LAML. We compared the expression of B4GALT1 in LAML and healthy samples using the Wilcoxon rank-sum test. Furthermore, the association between B4GALT1 and survival rates was investigated using Kaplan-Meier analysis and Cox regression. The nomogram obtained by Cox analysis predicts the effect of B4GALT1 on the prognosis. To assess B4GALT1-related genes' enrichment pathway and function and the correlation between B4GALT1 and immune features, GO/KEGG, protein-protein interaction network, and single sample gene set enrichment analysis were used. In addition, B4GALT1-specific siRNAs were used to verify the effect of B4GALT1 on apoptosis. The results showed that B4GALT1 is overexpressed in LAML and has some reference value in the diagnostic and prognostic assessment of LAML. Moreover, functional enrichment showed that B4GALT1 and its 63 associated genes were closely associated with the negative regulation of the apoptotic signaling pathway. Silencing B4GALT1 significantly promoted apoptosis. In addition, B4GALT1 expression was positively correlated with the infiltration levels of macrophages, regulatory T-cell (Tregs), and Th17 cells; in contrast, B4GALT1 expression was negatively correlated with the infiltration levels of T helper cells, Mast cells, and NK cells. In conclusion, our study shows that B4GALT1 may play a vital role in the occurrence of LAML.

Mannan-Binding Lectin Regulates the Th17/Treg Axis Through JAK/STAT and TGF-ß/SMAD Signaling Against Candida albicans Infection.

Background: Mannan-binding lectin (MBL) is a key molecule in innate immunity and activates the lectin complement pathway, which plays an important role in resisting Candida albicans (C. albicans) infection. However, the underlying mechanism of this resistance to infection remains unclear. Methods: In this study, we investigated how MBL regulates the differentiation of CD4+ T cells into T helper type 17 (Th17) and T regulatory (Treg) cells against C. albicans in mice, as well as the underlying mechanisms. We generated MBL double-knockout (KO) mice and infected them with C. albicans by intraperitoneal injection. Results: Compared with that in wild-type (WT) mice, the percentage of Th17 cells increased in MBL-null mice, whereas Treg cells decreased, indicating that MBL might regulate the Th17/Treg balance. In addition, in MBL-null mice, the expression levels of interleukin (IL)-17A, IL-21, and the master transcription factor of Th17 cells, RORγt, significantly increased. Conversely, IL-10, IL-2, and the Treg-specific transcription factor, Foxp3, decreased. Moreover, we found that the levels of TGF-ß and IL-6 upregulated in MBL-null mice. Mechanistically, we found that MBL regulated the TGF-ß/SMAD pathway through the inhibition of p-SMAD2 and promotion of p-SMAD3, and mediated the JAK/STAT pathway through the inhibition of p-JAK2 and p-STAT3 and promotion of p-JAK3 and p-STAT5. MBL double-KO mice showed a more severe inflammatory response and significantly lower survival rates with C. albicans infection. Conclusion: These results suggest that MBL regulates the Th17/Treg cell balance to inhibit inflammatory responses, possibly via IL-6- and TGF-ß-mediated JAK/STAT and TGF-ß/SMAD signaling, and play an important role in anti-C. albicans infection.

Sulforaphane regulates the proliferation of leukemia stem-like cells via Sonic Hedgehog signaling pathway.

Sulforaphane (SFN), the main ingredient in broccoli/broccoli sprouts, has a good anticancer effect in a wide variety of tumors, but whether SFN affects acute leukemia is not elucidated. Due to the self-renewal capability for leukemia stem cells, acute leukemia has a high relapse rate. This study explored the effects and related molecular mechanisms of SFN on the proliferation of leukemia stem-like cells in acute myeloid leukemia cells. We found that SFN inhibited the proliferation of leukemia stem-like cells in vitro and in vivo. Meanwhile, we observed that SFN could regulate the stem characteristic of leukemia cells. After SFN treatment, the expression of the key players in the Sonic Hedgehog (Shh) signaling pathway was significantly decreased at the transcriptional and protein levels. To further determine the contribution of the Shh signaling molecular mechanism to SFN-mediated self-renewal capability of LSCs, we then manipulated the Shh gene in the leukemia cells to either overexpress the gene using lentiviral vector transduction or knockdown the gene via siRNA. The results demonstrated that SFN suppressed proliferation in Shh-overexpressed cells more than in Shh-downregulated cells, suggesting that SFN negatively modulates proliferation of leukemia stem-like cells via affecting the Shh signaling pathway. Altogether, these results suggest that SFN is a potent anti-leukemia agent that has inhibitory effects on leukemia stem-like cells' proliferation by regulating the Shh signaling pathway.

[Effect and Mechanism of Sulforaphane on G2/M Phase Arrest of Acute Myeloid Leukemia KG1a and KG1 Cells].

OBJECTIVE: To investigate the effect of sulforaphane (SFN) on G2/M phase arrest of acute myeloid leukemia cells and its molecular mechanism. METHODS: KG1a and KG1cells were treated by different concentrations of SFN for 48 h. Flow cytometry (FCM) was used to analyze the phase distribution of cell cycle. High-throughput sequencing was used to detect the effect of SFN on the expression of cell cycle related genes in KG1a cells. The mRNA expression of P53, P21, CDC2 and CyclinB1 were detected by qPCR. The protein expression of P53, CDC2, P-CDC2 and CyclinB1 were detected by Western blot. RESULTS: Cells in the G2/M phase were increased from 11.9% to 54.0% in KG1a cells and 18.5% to 83.3% in KG1 cells after treated by SFN (8 µ mol / L) for 48 hours（P<0.001）. KEGG analysis indicated that P53 pathway was enriched in KG1a cells after treated by SFN. The heat-map graph showed that SFN could change the relevant genes of the cell cycle in KG1a cells. After SFN treatment, the mRNA level of P53 and P21 were significantly increased in KG1 and KG1a cells（P<0.05 or P<0.01）. The mRNA level of CDC2 showed a decrease trend with the increasing dose of SFN. At the dosage of 8 µmol /L, the mRNA expression levels of CDC2 was significantly lower than that in control group（P<0.05）. At the same time, the protein level of P53 was significantly increased in KG1 and kG1a cells after treated by SFN（P<0.05）. The protein level of CDC2 showed a decrease trend with the increasing dose of SFN in a dose manner（r=0.9482 and r=0.8977）. The protein levels of CDC2 in SFN 8 and 12 µ mol/L groups were significantly lower than that in control group(P<0.05, P<0.01). The protein levels of P-CDC2 was increased. But the change of mRNA and protein level of CyclinB1 was not significant. CONCLUSION: SFN induces leukemia cells to block in G2/M phase by activating P53 signaling pathway, which can inhibit the expression of CDC2 and the activity of CDC2/cyclinB1.

Genetic Expression Screening of Arsenic Trioxide-Induced Cytotoxicity in KG-1a Cells Based on Bioinformatics Technology.

Acute myeloid leukemia (AML) is a malignant tumor of the hematopoietic system, and leukemia stem cells are responsible for AML chemoresistance and relapse. KG-1a cell is considered a leukemia stem cell-enriched cell line, which is resistant to chemotherapy. Arsenic trioxide (ATO) is effective against acute promyelocytic leukemia as a first-line treatment agent, even as remission induction of relapsed cases. ATO has a cytotoxic effect on KG-1a cells, but the mechanism remains unclear. Our results demonstrated that ATO can inhibit cell proliferation, induce apoptosis, and arrest KG-1a cells in the G2/M phase. Using transcriptome analysis, we investigated the candidate target genes regulated by ATO in KG-1a cells. The expression profile analysis showed that the ATO had significantly changed gene expression related to proliferation, apoptosis, and cell cycle. Moreover, MYC, PCNA, and MCM7 were identified as crucial hub genes through protein-protein interaction network analysis; meanwhile, the expressions of them in both RNA and protein levels are down-regulated as confirmed by quantitative polymerase chain reaction and Western blot. Thus, our study suggests that ATO not only inhibits the expression of MYC, PCNA, and MCM7 but also leads to cell cycle arrest and apoptosis in KG-1a cells. Overall, this study provided reliable clues for improving the ATO efficacy in AML.

Sulforaphane inhibits self-renewal of lung cancer stem cells through the modulation of sonic Hedgehog signaling pathway and polyhomeotic homolog 3.

Sulforaphane (SFN), an active compound in cruciferous vegetables, has been characterized by its antiproliferative capacity. We investigated the role and molecular mechanism through which SFN regulates proliferation and self-renewal of lung cancer stem cells. CD133+ cells were isolated with MACs from lung cancer A549 and H460 cells. In this study, we found that SFN inhibited the proliferation of lung cancer cells and self-renewal of lung cancer stem cells simultaneously. Meanwhile, the mRNA and protein expressions of Shh, Smo, Gli1 and PHC3 were highly activated in CD133+ lung cancer cells. Compared with siRNA-control group, Knock-down of Shh inhibited proliferation of CD133+ lung cancer cells, and decreased the protein expression of PHC3 in CD133+ lung cancer cells. Knock-down of PHC3 also affected the proliferation and decreased the Shh expression level in CD133+ lung cancer cells. In addition, SFN inhibited the activities of Shh, Smo, Gli1 and PHC3 in CD133+ lung cancer cells. Furthermore, the inhibitory effect of SFN on the proliferation of siRNA-Shh and siRNA-PHC3 cells was weaker than that on the proliferation of siRNA-control cells. Sonic Hedgehog signaling pathway might undergo a cross-talk with PHC3 in self-renewal of lung cancer stem cells. SFN might be an effective new drug which could inhibit self-renewal of lung cancer stem cells through the modulation of Sonic Hedgehog signaling pathways and PHC3. This study could provide a novel way to improve therapeutic efficacy for lung cancer stem cells.

The responses of the gut microbiota to MBL deficiency.

Mannose-binding lectin (MBL) deficiency is a common innate immune system deficiency, and is associated with exacerbations and increased colonization of some pathogens. However, the response of the gut microbiota, a pivotal factor in host health, to MBL deficiency is not clear. In this study, MBL-/- and wild-type (WT) mice were generated by backcrossing from MBL-A and MBL-C knockout (KO) mice, and fecal samples were collected at different ages (4th, 8th, 12th, 19th and 27th weeks). The gut microbiota was analyzed by high-throughput sequencing with universal 16S rDNA primers (V3-V5 region). The results showed that structural segregation of the gut microbiota occurred at the 8th, 12th, 19th and 27th weeks of age, although there were no significant differences in alpha diversities between MBL-/- and WT mice at different ages. Impressively, in MBL-/- mice, Akkermansia (from the family Verrucomicrobiaceae) were decreased significantly, Lactobacillus (from the family Lactobacillaceae) abundances, Alistipes and Rikenella (both from the family of Rikenellaceae) were always enriched. Network analysis showed that more interactions existed in the gut microbiota from WT mice (33 nodes and 70 edges) than in the gut microbiota from MBL-/- mice (23 nodes and 40 edges). The 16S rDNA function prediction results indicated that the abundances of predicted genes in the "immune system disease", "metabolic disease" and "nucleotide metabolism" pathways were significantly increased in the MBL-/- mice. In conclusion, this study revealed that the gut microbiota changed in MBL deficient mice, especially at ages older than 4 weeks.

MicroRNA-143-3p suppresses tumorigenesis by targeting catenin-δ1 in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third most common malignancy in the United States. Mounting microRNAs (miRNAs) have been identified as oncogenes or tumor suppressors in various cancers including CRC. MATERIALS AND METHODS: The levels of microRNA-143-3p (miR-143-3p) and catenin-δ1 (CTNND1) were determined by RT-qPCR assay. Cell proliferative ability was assessed by Cell Counting Kit-8 assay. Cell migratory and invasive capacities were measured by transwell migration and invasion assay. Luciferase reporter assay was conducted to explore whether miR-143-3p could bind with CTNND1 3'UTR. CTNND1 protein level was determined through Western blot assay. Mouse xenograft models of CRC were established to test the functions and molecular basis of miR-143-3p in the development of CRC in vivo. RESULTS: Low amounts of miR-143-3p were expressed in CRC tissues and cells. Functional analysis revealed that miR-143-3p overexpression suppressed cell proliferation, migration and invasion in CRC. Molecular mechanism exploration indicated that miR-143-3p directly targeted CTNND1. Moreover, enforced expression of CTNND1 contributed to cell proliferation, migration and invasion in CRC, and CTNND1 silencing exerted opposite effects. Restoration experiments disclosed that CTNND1 upregulation weakened the inhibitory effects of miR-143-3p on CRC cell proliferation, migration and invasion. Additionally, miR-143-3p inhibited the growth of HCT116-derived xenograft tumors by targeting CTNND1 in vivo. CONCLUSION: miR-143-3p hampered the development and progression of CRC by targeting CTNND1 in vitro and in vivo, deepening our understanding of the functions and molecular basis of miR-143-3p in the tumorigenesis of CRC and providing some candidate prognostic markers or therapeutic targets for CRC.

Mannan-Binding Lectin Suppresses Peptidoglycan-Induced TLR2 Activation and Inflammatory Responses.

Peptidoglycan (PGN), as the major components of the bacterial cell wall, is known to cause excessive proinflammatory cytokine production. Toll-like receptor 2 (TLR2) is abundantly expressed on immune cells and has been shown to be involved in PGN-induced signaling. Although more and more evidences have indicated that PGN is recognized by TLR2, the role of TLR2 PGN recognition is controversial. Mannan-binding lectin (MBL), a plasma C-type lectin, plays a key role in innate immunity. More and more evidences show that MBL could suppress the amplification of inflammatory signals. Whether MBL can alter PGN-elicited cellular responses through TLR2 in macrophages is still unknown, and possible mechanism underlying it should be investigated. In this study, we found that MBL significantly attenuated PGN-induced inflammatory cytokine production, including TNF-α and IL-6, in PMA-stimulated THP-1 cells at both mRNA and protein levels. The expression of TLR2 was strongly induced by PGN stimulation. Furthermore, the administration of TLR2-neutralized antibody effectively suppressed PGN-induced TNF-α and IL-6 expression. These results supplied the evidence that PGN from Saccharomyces cerevisiae could be recognized by TLR2. In addition, we also found that MBL decreased PGN-induced TLR2 expression and suppressed TLR2-mediated downstream signaling, including the phosphorylation of IκBα, nuclear translocation of NF-κBp65, and phosphorylation of MAPK p38 and ERK1/2. Administration of MBL alone did not have an effect on the expression of TLR2. Finally, our data showed that PGN-mediated immune responses were more severely suppressed by preincubation with MBL and indicated that MBL can combine with both TLR2 and PGN to block the inflammation cytokine expression induced by PGN. All these data suggest that MBL could downregulate inflammation by modulating PGN/TLR2 signaling pathways. This study supports an important role for MBL in immune regulation and signaling pathways involved in inflammatory responses.

Berberine attenuate staphylococcal enterotoxin B-mediated acute liver injury via regulating HDAC expression.

Staphylococcal enterotoxin B (SEB) has been documented to be implicated in the pathogenesis of liver injury in the experimental models of hepatitis. However, the underlying mechanism of SEB-induced acute liver injury (ALI) remains to be further explored. In our study, we explored the therapeutic effectiveness of berberine (BBR), a natural isoquinoline alkaloid, in the SEB-induced ALI. In our study, we found that injection of SEB into D-galactosamine (D-gal)-sensitized mice induced ALI, as demonstrated by an increase of levels of alanine aminotransferase and aspartate aminotransferase, massive infiltration of immune cells into the liver, and pro-inflammatory cytokine release. However, intragastric administration of BBR attenuated SEB-induced ALI in mice. Meanwhile, we discovered that BBR treatment suppressed activation of splenocytes and pro-inflammatory cytokine release in SEB-stimulated splenocytes. Moreover, mechanistic analyses demonstrated that BBR was effective at inhibiting the expression of class I HDAC, but not class II, in SEB-stimulated splenocytes. Furthermore, trichostatin A, a standard HDAC inhibitor, alleviated activation of splenocytes and pro-inflammatory cytokine release in SEB-stimulated splenocytes. Taken together, we inferred from these results that BBR attenuated SEB-mediated ALI through repressing the class I HDAC enzyme, suggesting that BBR may constitute a novel therapeutic modality to prevent SEB-mediated inflammation and ALI.

Sulforaphane reverses gefitinib tolerance in human lung cancer cells via modulation of sonic hedgehog signaling.

Gefitinib is a targeted anticancer drug that was developed as an effective clinical therapy for lung cancer. Numerous patients develop gefitinib resistance in response to treatment. Sulforaphane (SFN) is present in cruciferous vegetables, and has been demonstrated to inhibit the malignant growth of various types of cancer cells. To investigate the role of SFN in gefitinib resistance, a gefitinib-tolerant PC9 (PC9GT) cell model was established by continually exposing PC9 cells to gefitinib. Cell viability was measured using a cell proliferation assay. Components of the sonic hedgehog (SHH) signaling pathway and markers of lung cancer stem cells were detected via western blotting. SFN markedly inhibited the proliferation of PC9GT and PC9 cells in a dose-dependent manner; combination SFN/gefitinib treatment also markedly decreased PC9GT cell proliferation, compared with SFN or gefitinib administered alone (P<0.05). Western blot analysis revealed that the expression of SHH, Smoothened (SMO), zinc finger protein GLI1 (GLI1), GLI2, CD133 and CD44 were upregulated in PC9GT cells, as compared with in PC9 cells. Furthermore, SFN markedly inhibited the expression of SHH, SMO and GLI1 in PC9GT and PC9 cells in a dose dependent manner, and SFN combined with gefitinib markedly inhibited the expression of SHH, SMO, GLI1, CD133 and CD44 in PC9GT cells when compared with SFN or gefitinib monotherapy. The results of the present study demonstrated that SFN inhibits the proliferation of gefitinib-tolerant lung cancer cells via modulation of the SHH signaling pathway. Therefore, combined SFN and gefitinib therapy may be an effective approach for the treatment of lung cancer.

[Effects of Arsenic Trioxide on K562 Cell Proliferation and Its Mechanisms].

OBJECTIVE: To investigate the effects of arsenic trioxide (As2O3) on K562 cell proliferation by regulating cell cycle protein D1 and cyclin-dependent kinase inhibitor p27kip1. METHODS: MTT was used to detect the effect of As2O3 on K562 cell proliferation, so as to screen out the appropriate drug concentration. Furthermore, the K562 cell apoptosis was observed by microscopy. The expression of CyclinD1 and p27kip1 in K562 cells treated with As2O3 was analyzed by reverse transcription-polymerase chain reaction(RT-PCR), immunohistochemistry and Western blot. RESULTS: As2O3 could inhibit the proliferation of K562 cells in a dose- and time- dependent manner (r= 0.967). And the apoptosis cell number in As2O3 group was significantly higher than that in the control group(P<0.05). As2O3 could markedly inhibit the expression of CyclinD1 in K562 cells(P<0.05), but the expression of P27kip1 was not significantly changed after As2O3 treatment. CONCLUSIONS: As2O3 can induce K562 cell apoptosis and inhibit K562 cell proliferation by regulating the expression of CyclinD1.

[Mannan-Binding Lectin Inhibits Candida Albicans-Induced DC Maturation and Cytokine Secretion].

OBJECTIVE: To investigate the effects of mannan-binding lectin (MBL) on the maturation and cytokine secretion of human dendritic cells (DC) induced by Candida albicans (C. albicans). METHODS: The plastic-adherent mononuclear cells were prepared from the blood of healthy adult volunteers. The human peripheral blood mononuclear cells-derived dendritic cells (MNC-DC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4, and then cultured for 2 days in presence or absence of C. albicans at varying concentration of human MBL ranging from 1 to 20 mg/L. DC's shape and characters were observed under inverted microscopy, the expression of CD83 and CD86 on DC was analyzed by FACS. The levels of TNF-α and IL-6 were detected by ELISA. FACS also was used to investigate the interaction of MBL with immature DC(imDC) and C. albicans. Western blot was used to detect C. albicans-induced IκBα phosphorylation and p65/NF-κB translocation in DC. RESULTS: MBL at higher concentrations (10-20 mg/L) down-regulated the expression of CD83 and CD86 on the monocyte-derived dentritic cells(MoDC) induced by C. albicans, and inhibited the production of TNF-α and IL-6 induced by C. albicans. FACS showed that MBL could not only bind to C. albicans but also bind to imDCs in a Ca2+-dependent manner. Western blot showed that MBL could decrease the phosphorylation of IκBα and the nuclear translocation of p65/ NF-κB. CONCLUSION: MBL may inhibit TNF-α and IL-6 production induced by C. albicans in DC through NF-κB signaling pathways, suggesting that MBL can play some roles in the regulation of C. albicans-induced immune response.

[Mechanism of MBL inhibiting the LPS-induced DC maturation].

The study was aimed to investigate the mechanism of mannan-binding lectin (MBL) on bacterial lipopolysaccharide (LPS)-induced human peripheral blood monocyte-derived dendritic cell (DC) maturation. The monocytes were prepared from the peripheral blood of healthy adult volunteers. The immature dendritic cells (imDC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4. FACS was used to investigate the interaction of MBL with imDC and the impact of MBL on LPS binding to imDC. ELISA and Western blot was used to analyze the interaction of MBL with soluble TLR4 ectodomain protein (sTLR4); Western blot was used to detect LPS-induced NF-κB translocation in imDC. The results showed that MBL could directly bind to imDC in the presence of calcium. sTLR4 protein or LPS could competitively inhibit the binding of MBL to imDC. ELISA and Western blot showed that MBL could evidently bind to sTLR4 protein in a concentration-dependent manner. FACS showed that MBL could competitively inhibit the binding of LPS to imDC by binding to imDC directly. Western blot showed that MBL decreased LPS-induced NF-κB translocation in imDC. It is concluded that MBL may competitively inhibit the binding of LPS to imDC by binding to TLR4 expressed on imDC, resulted in inhibition of LPS-induced DC maturation, suggesting that MBL can regulate DC maturation through ligand-binding. This study provides the good foundation to clarify the mechanism of MBL inhibiting the LPS-induced DC maturation.

Mannan-binding lectin inhibits Candida albicans-induced cellular responses in PMA-activated THP-1 cells through Toll-like receptor 2 and Toll-like receptor 4.

BACKGROUND: Candida albicans (C. albicans), the most common human fungal pathogen, can cause fatal systemic infections under certain circumstances. Mannan-binding lectin (MBL),a member of the collectin family in the C-type lectin superfamily, is an important serum component associated with innate immunity. Toll-like receptors (TLRs) are expressed extensively, and have been shown to be involved in C. albicans-induced cellular responses. We first examined whether MBL modulated heat-killed (HK) C. albicans-induced cellular responses in phorbol 12-myristate 13-acetate (PMA)-activated human THP-1 macrophages. We then investigated the possible mechanisms of its inhibitory effect. METHODOLOGY/PRINCIPAL FINDING: Enzyme-linked immunosorbent assay (ELISA) and reverse transcriptasepolymerase chain reaction (RT-PCR) analysis showed that MBL at higher concentrations (10-20 µg/ml) significantly attenuated C. albicans-induced chemokine (e.g., IL-8) and proinflammatory cytokine (e.g., TNF-α) production from PMA-activated THP-1 cells at both protein and mRNA levels. Electrophoretic mobility shift assay (EMSA) and Western blot (WB) analysis showed that MBL could inhibit C. albicans-induced nuclear factor-κB (NF-κB) DNA binding and its translocation in PMA-activated THP-1 cells. MBL could directly bind to PMA-activated THP-1 cells in the presence of Ca(2+), and this binding decreased TLR2 and TLR4 expressions in C. albicans-induced THP-1 macrophages. Furthermore, the binding could be partially inhibited by both anti-TLR2 monoclonal antibody (clone TL2.1) and anti-TLR4 monoclonal antibody (clone HTA125). In addition, co-immunoprecipitation experiments and microtiter wells assay showed that MBL could directly bind to the recombinant soluble form of extracellular TLR2 domain (sTLR2) and sTLR4. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that MBL can affect proinflammatory cytokine and chemokine expressions by modifying C. albicans-/TLR-signaling pathways. This study supports an important role for MBL on the regulation of C. albicans-induced cellular responses.